BR112021011961A2 - COMPOSITIONS COMPRISING POLYSACCHARIDE-PROTEIN CONJUGATES FROM STREPTOCOCCUS PNEUMONIAE AND METHODS OF USE THEREOF - Google Patents

Abstract

compositions comprising streptococcus pneumoniae polysaccharide-protein conjugates and methods of using the same. the present invention relates to multivalent immunogenic compositions comprising more than one s.polysaccharide-protein conjugate. pneumoniae, wherein each of the conjugates comprises a polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, in which s. pneumoniae are as defined in the present invention. in some embodiments, at least one of the polysaccharide-protein conjugates is formed by a conjugation reaction comprising an aprotic solvent. in additional embodiments, each of the polysaccharide-protein conjugates is formed by a conjugation reaction comprising an aprotic solvent. Also provided are methods of inducing a protective immune response in a human patient comprising administering the multivalent immunogenic compositions of the present invention to the patient. multivalent immunogenic compositions are useful in providing protection against S infection. pneumoniae and/or pneumococcal diseases caused by s. pneumoniae. the compositions of the present invention are also useful as part of treatment regimens that provide complementary protection for patients who have been vaccinated with a multivalent vaccine indicated for the prevention of pneumococcal disease.

Description

COMPOSITIONS COMPRISING POLYSACCHARIDE CONJUGATE- STREPTOCOCCUS PNEUMONIAE PROTEIN AND METHODS OF USE OF SAME

FIELD OF THE INVENTION The present invention provides multivalent immunogenic compositions that have distinct polysaccharide-protein conjugates. Each conjugate consists of a capsular polysaccharide prepared from a different Streptococcus pneumoniae serotype conjugated to a carrier protein, preferably CRM197. Immunogenic compositions provide broad coverage against pneumococcal disease. The present application contains a listing of sequences which has been submitted electronically in ASCII format and which is incorporated herein by reference in its entirety. Said ASCII copy, created on December 3, 2019, is named 24683WOPCT-SEQTXT-03DEC2019 and is 6 kilobits in size.

BACKGROUND OF THE INVENTION Streptococcus pneumoniae is a gram-positive bacterium and the most common cause of invasive bacterial disease (such as pneumonia, bacteremia, meningitis and otitis media) in neonates and young children. Pneumococcal is encapsulated with a chemically bound polysaccharide that confers serotype specificity. There are more than 90 known pneumococcal serotypes, and the capsule is the main virulence determinant for pneumococcal disease, as the capsule not only protects the inner surface of bacteria against complement, but the complement itself is immunogenically poor. Polysaccharides are T cell-independent antigens, and in most cases, they may not be processed or present in MHC molecules to interact with T cells. They may, however, stimulate the immune system through an alternative mechanism. involving cross-linking of surface receptors on B cells.

Multivalent pneumococcal polysaccharide vaccines that have been licensed for many years have proven valuable in preventing pneumococcal disease in adults, particularly the elderly and those at high risk.

However, neonates and young children responded poorly to unconjugated pneumococcal polysaccharides.

The pneumococcal conjugate vaccine, Prevnar®, which contains the 7 most frequently isolated serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) that cause invasive pneumococcal disease in young children and neonates at this time, has been first licensed in the United States in February 2000. After the universal use of Prevnar® in the United States, there was a significant reduction in invasive pneumococcal disease in children due to the serotypes present in Prevnar®. See Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 2005, 54(36): 893 to 897. However, there are limitations in serotype coverage with Prevnar® in certain regions of the world and some evidence of certain serotypes emerging in the world. United States (eg 19A and others). See O'Brien et al., 2004, Am J Epidemiol 159: 634 to 644; Whitney et al., 2003, N Engl J Med 348: 1737 to 1746; Kyaw et al., 2006, N Engl J Med 354: 1455 to 1463; Hicks et al., 2007, J Infect Dis 196: 1346 to 1354; Traore et al., 2009, Clin Infect Dis 48: S181 to S189. US Patent Application Publication 2006/0228380 describes a 13-valent pneumococcal polysaccharide-protein conjugate vaccine including serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.

Chinese Patent Application Publication CN 101590224 A describes a 14-valent pneumococcal polysaccharide-protein conjugate vaccine including serotypes 1, 2, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F and 23F.

Other PCVs encompassed 7, 10, 11, or 13 of the serotypes contained in PCV-15 (US Publication 2011/0195086), but immunological interference was observed for some serotypes (eg, inferior protection for serotype 3 in GSK PCV-11) and lower response rates for serotype 6B in Pfizer's PCV-13 (PREVNAR® 13). See Prymula et al., 2006, Lancet 367: 740 to 748 and Kieninger et al., Safety and Immunologic Non-inferiority of 13-valent Pneumococcal Conjugate Vaccine Compared to 7-valent Pneumococcal Conjugate Vaccine Given as a 4-Dose Series in Healthy Infants and Toddlers, presented at the 48th ICAAC/ISDA Annual, 46th Annual Meeting, Washington Capital, October 25-28, 2008. Current multivalent pneumococcal vaccines have been effective in reducing the incidence of pneumococcal disease associated with those serotypes present in the vaccines. However, the prevalence of serotypes that express pneumococcal strains not present in currently available vaccines has been increasing. Consequently, there is a need for additional pneumococcal vaccine compositions that can provide protection against pneumococcal serotypes not present in currently available vaccines.

SUMMARY OF THE INVENTION The present invention provides multivalent immunogenic compositions comprising polysaccharide-protein conjugates of S. pneumoniae, wherein each of the conjugates comprises a polysaccharide of a serotype of S. pneumoniae conjugated to a carrier protein, and wherein the polysaccharide conjugates -protein include polysaccharides from a group of S. pneumoniae serotypes selected from the group consisting of: a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,

23F, 24F, 33F and 35B; p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; q) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; s) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; and aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

The present invention provides a multivalent immunogenic composition comprising 22 distinct polysaccharide-protein conjugates, each of the conjugates comprising a capsular polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, wherein the polysaccharides are prepared from serotypes from S. pneumoniae 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

The present invention provides a multivalent immunogenic composition comprising 22 distinct polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, and wherein the polysaccharide-protein conjugates include polysaccharides from a group of S. pneumoniae serotypes selected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

The present invention provides a multivalent immunogenic composition comprising 23 distinct polysaccharide-protein conjugates, each of the conjugates comprising a capsular polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, wherein the polysaccharides are prepared from serotypes from S. pneumoniae 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

The present invention provides a multivalent immunogenic composition comprising 23 distinct S. pneumoniae protein-polysaccharide conjugates, wherein each of the conjugates comprises a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein each polysaccharide conjugate -distinct protein comprises a polysaccharide from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B,

respectively, and wherein the carrier protein is CRM197. The present invention provides a multivalent immunogenic composition comprising 24 distinct polysaccharide-protein conjugates, each of the conjugates comprising a capsular polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, wherein the polysaccharides are prepared from serotypes pneumoniae 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

The multivalent immunogenic composition comprising 24 distinct S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein each distinct polysaccharide-protein conjugate comprises a polysaccharide of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and wherein the carrier protein is CRM197. The present invention provides a multivalent immunogenic composition comprising 24 distinct polysaccharide-protein conjugates, each of the conjugates comprising a capsular polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, wherein the polysaccharides are prepared from serotypes pneumoniae 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

The present invention provides a multivalent immunogenic composition comprising 24 distinct S. pneumoniae protein-polysaccharide conjugates, wherein each of the conjugates comprises a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein each polysaccharide conjugate -distinct protein comprises a polysaccharide from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and wherein the carrier protein is CRM197. The present invention provides a multivalent immunogenic composition comprising up to 33 distinct polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, and wherein the polysaccharide-protein conjugates include polysaccharides from a group of S. pneumoniae serotypes selected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C , 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, including additionally one, two, three, four, five, six, seven, eight or nine additional S. pneumoniae serotypes selected from 7C, 9N, 16F, 21, 23A, 31, 34, 35F and 38. The present invention provides a multivalent immunogenic composition comprising up to 30 distinct polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, and in which the co Polysaccharide-protein conjugates include polysaccharides from a group of S. pneumoniae serotypes selected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14 , 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, including additionally one, two, three, four, five or six additional S. pneumoniae serotypes selected from 7C, 9N, 16F, 23A, 35F and 38. The present invention provides a multivalent immunogenic composition comprising up to 33 distinct polysaccharide-protein conjugates, each of the conjugates comprising a polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, and in that the polysaccharide-protein conjugates include polysaccharides from a group of S. pneumoniae serotypes selected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F , 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, additionally including one, two, three, four, five , six, seven, eight or nine additional S. pneumoniae serotypes selected from 7C, 9N, 16F, 21, 23A, 31, 34, 35F and 38. The present invention provides a multivalent immunogenic composition comprising up to 30 polysaccharide conjugates -protein, wherein each of the conjugates comprises a polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, and wherein the polysaccharide-protein conjugates include polysaccharides from a group of S. pneumoniae serotypes selected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, including additionally one, two, three, four, five or six additional S. pneumoniae serotypes selected from 7C, 9N, 16F, 23A, 35F and 38. In some embodiments, at least one of the polysaccharide conjugates -protein is formed by a conjugation reaction comprising an aprotic solvent, e.g. dimethyl 1sulfoxide (DMSO). In specific embodiments, each of the polysaccharide-protein conjugates is formed by a conjugation reaction comprising an aprotic solvent, eg (DMSO). Also provided are methods for inducing a protective immune response in a human patient comprising administering the multivalent immunogenic compositions of the present invention to the patient.

In some embodiments of the methods of the present invention, the patient has previously been treated with a multivalent pneumococcal vaccine. The multivalent immunogenic composition of the present invention can be used as part of a treatment regimen with a different complementary pneumococcal vaccine. Accordingly, the present invention provides a method of inducing a protective immune response in a human patient comprising administering a multivalent immunogenic composition of the present invention to the patient, further comprising administering a multivalent pneumococcal vaccine to the patient in any order. In particular embodiments, the multivalent pneumococcal vaccine is comprised of multiple S. pneumoniae protein-polysaccharide conjugates wherein each of the conjugates comprises polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein. In other embodiments, the multivalent pneumococcal vaccine is comprised of unconjugated capsular polysaccharides. The present invention also provides multivalent immunogenic compositions comprising polysaccharide-protein conjugates of S. pneumoniae wherein each of the conjugates comprises a polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes provide reaction. cross to other selected serotypes.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1. Pre-immunization (Pre), post-dose 1 (PD1), 2 (PD2) and 3 (PD3) IgG antibody dilution titers as determined by ECL for PCV22-immunized mice without adjuvant (PCV22 without adj) or formulated with aluminum phosphate adjuvant (PCV22/APA). Reading from left to right; Pre PCV22 without adj, Pre PCV22/APA, PD1 PCV22 without adj, PD1 PCV22/APA, PD2 PCV22 without adj, PD2 PCV22/APA, PD3 PCV22 without adj, and PD3

PCV22/APA.

FIGURE 2. PCV22/APA ECL dilution titrator ratio compared to PCV22 without adjuvant (PCV22 without adj) at PD3. FIGURE 3. Titers of serotype-specific-specific (pre-immune, PD1, PD2, PD3) OPA dilution for mice immunized with PCV22 without adjuvant (PCV22 without adj) or formulated with APA (PCV22/APA). Reading from left to right; Pre PCV22 without adj, Pre PCV22/APA, PD1 PCV22 without adj, PD1 PCV22/APA, PD2 PCV22 without adj, PD2 PCV22/APA, PD3 PCV22 without adj, and PD3 PCV22/APA.

FIGURE 4. OPA dilution titrator ratio of PCV22/APA compared to PCV22 without adjuvant (PCV22 without adj) at PD3. FIGURE 5. PCV22 immunized mice are protected from intratracheal 24F challenge of S. pneumoniae.

FIGURE 6. Pre-immunization (Pre) IgG antibody dilution titer of PD1 and PD2 as determined by ECL for rabbits immunized with PCV22 without adjuvant or PCV22/APA.

The error bars represent the 95% confidence intervals (CI) of the geometric mean titrator (GMT). Reading from left to right; Pre PCV22 without adj, Pre PCV22/APA, PD1 PCV22 without adj, PD1 PCV22/APA, PD2 PCV22 without adj and PD2 PCV22/APA.

FIGURE 7. PCV22/APA ECL GMT ratio compared to PCV22 without PD2 adjuvant. Error bars represent 95% confidence intervals (CI). FIGURE 8. Serotype-specific-specific OPA dilution titrator (pre-immune “Pre” and PD2) for rabbits immunized with unadjuvanted PCV22 or PCV22/APA.

Error bars represent the change in functional antibody titers for five rabbits.

FIGURE 9. Pre-defined IgG antibody dilution titers.

(Pre), of PD1, PD2 and PD3 as determined by ECL for IRMs immunized with A) PCV23 without adjuvant, B) PCV23 (DMSO)/APA, C) PCV23(DMSO+Aq)/APA and D) PCV15/APA + PCV8/APA.

The error bars represent the 95% confidence intervals (CI) of the geometric mean titrator (GMT). FIGURE 10. Comparison of ECL antibody responses in IRMs (8 to 9 per group) following PCV23 vaccination with or without APA.

Symbols indicate ratios in A) PD1, B) PD2 or C) PD3. GMT ratios with error bars represent 95% CIs. FIGURE 11. Comparison of ECL antibody responses in IRMs (9 per group) following PCV23 (DMSO+Aq)/APA or co-administered PCV15/APA + PCV8/APA vaccination.

Symbols indicate ratios in A) PD1, B) PD2 and C) PD3. GMT ratios with error bars represent the 95% CIs. FIGURE 12. Comparison of ECL-driven antibody responses in IRMs (8 to 9 per group) following vaccination with unadjuvanted PCV23, PCV23(DMSO)/APA, PCV23(DMSO+Aq)/APA or PCV15/APA + PCV8/ APA.

A) PD1/Pre, B) PD2/Pre, C) PD3/Pre, D) PD2/PD1 and E) PD3/PD2. The symbols are GMT ratios with error bars representing the 95% CIs. FIGURE 13. (A) Pre-immuno (Pre), post-dose 1 (PD1) and 2 (PD2) IgG antibody dilution titers as determined by ECL for white New Zealand rabbits immunized with PCV24 formulated with phosphate adjuvant aluminum (PCV24/APA). The error bars represent the 95% confidence intervals (CI) of the geometric mean titrator (GMT). (B) Serotype-specific OPA dilution titrator (preimmune and PD2) for NZWRs immunized with PCV24/APA.

Error bars represent the change in functional antibody titers for eight NZWRs.

FIGURE 14. (A) Pre-IgM IgG Antibody Dilution Titers

immunization (Pre), post-dose 1 (PD1), 2 (PD2) and 3 (PD3) as determined by ECL for newborn rhesus monkeys (IRMs) immunized with PCV24 formulated with aluminum phosphate adjuvant (PCV24/APA ). The error bars represent the 95% confidence intervals (CI) of the geometric mean titrator (GMT). (B) Serotype-specific OPA dilution titrator (preimmune and PD3) for IRMs immunized with PCV24/APA. Error bars represent the change in functional antibody titers for five IRMs.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides multivalent immunogenic compositions comprising pneumococcal polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes are as defined in the present invention. In some embodiments the present invention provides multivalent immunogenic compositions comprising polysaccharide-protein conjugates from S. pneumoniae, wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, and wherein the polysaccharide conjugates -protein include polysaccharides from a group of S. pneumoniae serotypes selected from the group consisting of: a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; q) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F,

23B, 23F, 24F, 33F and 35B; r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; s) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; bb) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; cc) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; dd) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ff) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; gg) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; hh) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; kk) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ll) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; nn) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; oo) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; pp) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; qq) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; rr) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B,

23F, 24F, 33F, 35B and 39; ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; tt) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; uu) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; vv) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ww) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; zz) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; aa) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; and bb) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39. In In some embodiments, the multivalent immunogenic composition comprises pneumococcal serotypes selected from the group consisting of: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A , 19F, 22F, 23B, 23F, 24F, 33F and 35B; or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B ; or iv)

1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

In a particular embodiment, a multivalent immunogenic composition of the present invention comprises multiple pneumococcal S. pneumoniae protein-polysaccharide conjugates wherein each of the conjugates comprises a polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the serotypes pneumoniae comprise serotypes: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B .

Said compositions were found to be immunogenic in mice, rabbits and/or monkeys and generate functional antibody that killed vaccine-like bacterial strains at all doses tested.

The multivalent immunogenic compositions of the present invention are useful for immunizing a patient against vaccine-type S. pneumoniae serotypes and/or as part of a different complementary pneumococcal vaccine (or vaccines) treatment regimen. Accordingly, the present invention provides a method of inducing a protective immune response in a human patient comprising administering a multivalent immunogenic composition of the present invention to the patient, and further comprising administering a multivalent pneumococcal vaccine to the patient, in any order.

In other embodiments, the multivalent immunogenic compositions of the present invention are administered to a patient who has previously been immunized with a different multivalent pneumococcal vaccine.

In embodiments of the present invention, conjugates of at least one pneumococcal serotype are prepared using reductive amination in an aprotic solvent such as DMSO.

In additional embodiments, the multivalent immunogenic composition comprises pneumococcal conjugates which are each prepared using reductive amination in an aprotic solvent.

The use of DMSO solvent increases covalent polysaccharide-protein associations through direct consumption of lysine residues on the surface of the carrier protein.

The increased covalent association has a direct benefit in increasing the stability of the polysaccharide-protein conjugate of multivalent immunogenic compositions comprising DMSO-conjugated polysaccharide antigens.

I.

Definitions and Abbreviations As used throughout the specification and appended claims, the following abbreviations apply: APA aluminum phosphate adjuvant APC antigen presenting cell CI confidence interval DMSO dimethyl sulfoxide DS Polysaccharide-protein drug substance GMC geometric mean concentration GMT titrator geometric medium HPSEC high performance size exclusion chromatography IM intramuscularly or intramuscularly MRI newborn rhesus monkey LOS lipo-oligosaccharide LPS lipopolysaccharide MALS multi-angle light scattering MBC monovalent volume conjugate

Mn number average molecular weight MOPA multiplexed opsonophagocytic assays MW molecular weight NMWCO nominal molecular weight cutoff NZWR Rabbit New Zealand White OPA opsonophagocytosis assay PCV pneumococcal conjugate vaccine PD1 post-dose 1 PD2 post-dose 2 PD3 post-dose 3 PnPs Pneumococcal Polysaccharide Ps polysaccharide PS-20 polysorbate-20 RI UV refractory index UV w/v weight to volume In order that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below.

Unless specifically defined elsewhere herein, all other technical and scientific terms used in the present invention have the meaning as commonly understood by one skilled in the art to which this present invention pertains.

As used throughout the specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly states otherwise.

Reference to “or” indicates either or both possibilities, unless the context clearly indicates one of the indicated possibilities.

In some cases, “and/or” has been used to highlight either or both possibilities.

The terms "aqueous solvent" or "aqueous conditions" when used with conjugation, such as reductive amination, refer to the use of water, as the solvent for the conjugation reaction.

Water may contain buffers and other components except that no organic solvents are present.

The terms "aprotic solvent", "DMSO solvent" or "DMSO conditions" when used with conjugation, such as reductive amination, refer to the use of an aprotic solvent or a combination of aprotic solvents, (or DMSO, as applicable), as the solvent for the conjugation reaction.

The aprotic solvent may have some water present, for example up to 1%, 2%, 5%, 10% or 20%. The term "comprises" when used with the immunogenic composition of the present invention refers to the inclusion of any other components, such as adjuvants and excipients, or the addition of one or more polysaccharide-protein conjugates that are not specifically enumerated.

The term "consisting of" when used with the multivalent polysaccharide-protein conjugate mixture refers to a mixture having those particular S. pneumoniae polysaccharide-protein conjugates and no other S. pneumoniae polysaccharide-protein conjugates of a different serotype. "Consists essentially of" and variations such as "consists essentially of" or "consists essentially of", indicate the inclusion of any elements or group of elements cited, and the optional inclusion of other elements, of a similar or different nature to the elements cited, that do not materially alter the basic or novel properties of the specified dosage regimen, method or composition "Effective amount" of a composition of the present invention refers to a dose required to obtain antibodies that significantly reduce the likelihood or severity of infectivity of a microbe , for example, S. pneumoniae, during a subsequent challenge.

As used in the present invention, the phrase "indicated for the prevention of pneumococcal disease" means that a vaccine or immunogenic composition is approved by one or more regulatory authorities, such as the U.S. Food and Drug Administration, for the prophylaxis of one or more diseases caused by any serotype of S. pneumoniae, including, but not limited to: general pneumococcal disease, pneumococcal pneumonia, pneumococcal meningitis, pneumococcal bacteremia, invasive disease caused by S. pneumoniae, and otitis media caused by S. pneumoniae.

A "multivalent pneumococcal vaccine" is a pharmaceutical preparation comprising more than one active agent (eg, pneumococcal capsular polysaccharide or pneumococcal polysaccharide-protein conjugate) that provides active immunity to a disease or pathological condition caused by more than one S. pneumoniae serotype. .

The term "polysaccharide" is intended to include any antigenic saccharide element (or antigenic unit) commonly used in immunological and bacterial vaccine techniques, including, but not limited to, a "saccharide", an "oligosaccharide", a "polysaccharide", a "liposaccharide", a "lipooligosaccharide (LOS)", a "lipopolysaccharide (LPS)", a "glycosylate", a "glycoconjugate", and the like.

The term "non-adjuvanted", in the context of a vaccine or immunogenic composition of the present invention, means a pneumococcal polysaccharide composition, including, but not limited to, PCV8, PCV15, PCV22, PCV23 and PCV24, wherein the composition does not contain adjuvant. . "PCV8" refers to an immunogenic composition that contains polysaccharide from S. pneumoniae serotypes (PnPs) -8, -10A, -12F, -15A, -15C, -23B, -24F and -35B. “PCV15” refers to an immunogenic composition that contains polysaccharide from S. pneumoniae serotypes (PnPs) -1, -3, -4, -5, -6A, -6B, -7F, -9V, -14, -18C , -19A, -19F, -22F, -23F and -33F. “PCV22” refers to an immunogenic composition that contains polysaccharide from S. pneumoniae serotypes (PnPs) -1, -3, -4, -5, -6A, -6B, -7F, -9V, -10A, -12F , -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F and -35B. “PCV23” refers to an immunogenic composition that contains polysaccharide from S. pneumoniae serotypes (PnPs) -1, -3, -4, -5, -6A, -6B, -7F, -8, -9V, -10A , -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F and -35B. “PCV24” refers to an immunogenic composition that contains polysaccharide from S. pneumoniae serotypes (PnPs) -1, -3, -4, -5, -6A, -6B, -7F, -8, -9V, -10A , -11A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F and -35B. "CpG-containing nucleotide", "CpG-containing oligonucleotide", "CpG oligonucleotide", and similar terms refer to a nucleotide molecule 6 to 50 nucleotides in length that contains an unmethylated CpG chemical moiety.

See, for example, Wang et al., 2003, Vaccine 21:4297. CpG-containing oligonucleotides include oligonucleotides modified using any synthetic internucleoside linkages, modified base, and/or modified sugar.

An "adjuvant", as defined in the present invention, is a substance that serves to enhance the immunogenicity of an immunogenic composition of the present invention.

An immunological adjuvant may enhance an immune response to an antigen that is weakly immunogenic when administered alone, for example, by inducing no or weak antibody titers or cell-mediated immune response, increasing antibody titers to the antigen, and/or decreasing the dose of the antigen. effective in eliciting an immune response in the individual.

Thus, adjuvants are often provided to boost the immune response and are well known to those skilled in the art.

A "patient" (alternatively referred to herein as "subject") refers to a mammal capable of being infected with an S. pneumoniae.

In preferred embodiments, the patient is a human.

A patient can be treated prophylactically or therapeutically.

Prophylactic treatment provides sufficient protective immunity to reduce the likelihood or severity of a pneumococcal infection or the effects thereof, eg pneumococcal pneumonia.

Therapeutic treatment can be performed to reduce the severity or prevent recurrence of a S. pneumoniae infection or the clinical effects thereof.

Prophylactic treatment can be performed using a multivalent immunogenic composition of the present invention, as described in the present invention.

The composition of the present invention can be administered to the general population or to those persons at increased risk of pneumococcal infection, for example, the elderly, or those living with or caring for the elderly.

Those “in need of treatment” include those previously exposed to or infected with S. pneumoniae, those who have previously been vaccinated against S. pneumoniae, as well as those likely to have an infection, or anyone in whom a reduction in the likelihood of infection is desired. , for example, the immunocompromised, the elderly, children, adults or otherwise healthy individuals.

A "stable" multivalent immunogenic composition is a composition that has no significant changes observed at a refrigerated temperature (eg, 2 to 8 °C or 4 °C) for at least 1 month, 2 months, 3 months, 6 months, 12 months and/or 24 months.

Additionally, a "stable" composition includes one that exhibits desired characteristics at temperatures including 25°C and 37°C for periods including 1 month, 3 months, 6 months, 12 months and/or 24 months.

Typical acceptable criteria for stability are as follows: not more than about 5%, about 10%, about 15%, or about 20% variability in one or more of the following: (a) the number average molecular weight (Mn) of the S. pneumoniae protein-polysaccharide conjugates in the composition, (b) weight average molecular weight (Mw) of the S. pneumoniae protein-polysaccharide conjugates in the composition, (c) total polysaccharide concentration in the composition, ( d) maximum emission of the composition measured using intrinsic protein fluorescence spectroscopy at a particular excitation wavelength, e.g. 280 nanometers, and (e) the fluorescence intensity of the composition measured using intrinsic protein fluorescence spectroscopy in a particular excitation wavelength.

The term "stable" may also be used to refer to a particular pneumococcal conjugate in a multivalent immunogenic composition.

In such usage, the term refers to a conjugate that exhibits the desired properties over time, at a particular temperature, and such properties vary no more than about 5%, about 10%, about 15%, or about 20% over observed time and temperature.

II.

Multivalent Immunogenic Compositions The present invention provides multivalent immunogenic compositions comprising multiple polysaccharide-S. pneumoniae protein conjugates wherein each of the conjugates comprises a polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein.

Different aspects and modalities of the multivalent immunogenic compositions of the present invention are described, infra.

In one embodiment (Embodiment E1), the present invention provides a multivalent immunogenic composition comprising multiple S. pneumoniae protein-polysaccharide conjugates, each comprising capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise, consist or consist essentially of: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B or iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

In Modality E1 submodalities, the immunogenic composition does not comprise any S. pneumoniae polysaccharide-protein conjugates.

As used in the present invention, de-O-acetylated serotype 15B pneumococcal polysaccharide (DeOAc15B) is substantially equivalent to serotype 15C pneumococcal polysaccharide and has substantially identical NMR spectra (data not shown). As used in the present invention, de-O-acetylated serotype 15B pneumococcal polysaccharide and serotype 15C pneumococcal polysaccharide can each have an O-Acetyl content per repeat unit in the range of 0 to 5%, or in the range of 0 to 4%, or in the range of 0 to 3%, or in the range of 0 to 2%, or in the range of 0 to 1%, or in the range of 0 to 0.5%, or in the range of 0 to 0 .1% or in O-acetyl content.

In a report by Spencer B.L., et al., 15C pneumococcal polysaccharide may be slightly O-acetylated (Spencer, B.L. et al., Clin.

Vac.

Immune. (2017) 24(8): 1 to 13). Thus, in any of the embodiments of the multivalent immunogenic compositions in the present invention, de-O-acetylated serotype 15B (DeOAc15B) can be used in place of serotype 15C.

Processes for de-O-acetylation are known in the art, for example as described in Rajam et al., Clinical and Vaccine Immunology, 2007, 14(9):1223–1227. In certain embodiments of any of the multivalent immunogenic compositions of the present invention, including Embodiment E1 and any submodality thereof, the composition further comprises a pharmaceutically acceptable carrier.

Cross-Reactivity In one embodiment the present invention provides multivalent immunogenic compositions comprising polysaccharide-S. pneumoniae protein conjugates wherein each of the conjugates comprises a polysaccharide from a serotype of S. pneumoniae, including serotype 6C, conjugated to a carrier protein, in that S. pneumoniae serotype 6C cross-reacts against S. pneumoniae serotypes 6A and 6B.

In one embodiment the present invention provides multivalent immunogenic compositions comprising polysaccharide-protein conjugates from S. pneumoniae wherein each of the conjugates comprises a polysaccharide from a serotype of S. pneumoniae, including serotype 6A, conjugated to a carrier protein, in which serotype S. pneumoniae 6A provides cross-protection against S. pneumoniae serotypes 6B and/or 6C.

In one embodiment the present invention provides multivalent immunogenic compositions comprising polysaccharide-protein conjugates from S. pneumoniae wherein each of the conjugates comprises a polysaccharide from a serotype of S. pneumoniae, including serotype 6B, conjugated to a carrier protein, in which serotype S. pneumoniae 6B provides cross-protection against S. pneumoniae serotypes 6A and/or 6C.

In one embodiment the present invention provides multivalent immunogenic compositions comprising polysaccharide-protein conjugates of S. pneumoniae wherein each of the conjugates comprises a polysaccharide of a serotype of S. pneumoniae, including serotype 15C, conjugated to a carrier protein, wherein serotype S. pneumoniae 15C provides cross-protection against S. pneumoniae serotype 15B. In one embodiment the present invention provides multivalent immunogenic compositions comprising polysaccharide-protein conjugates from S. pneumoniae wherein each of the conjugates comprises a polysaccharide from a serotype of S. pneumoniae, including serotype 15B, conjugated to a carrier protein, wherein serotype S. pneumoniae 15B provides cross-protection against S. pneumoniae serotype 15C. Carrier Protein In particular embodiments of the present invention, CRM197 is used as the carrier protein. CRM197 is a non-toxic (i.e. toxoid) variant of diphtheria toxin that has the following amino acid sequence:

GADDVVDSSK SFVMENFSSY HGTKPGYVDS IQKGIQKPKS GTQGNYDDDW KEFYSTDNKY DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK VLALKVDNAE TIKKELGLSL TEPLMEQVGT EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI NNWEQAKALS VELEINFETR GKRGQDAMYE YMAQACAGNR VRRSVGSSLS CINLDWDVIR DKTKTKIESL KEHGPIKNKM SESPNKTVSE EKAKQYLEEF HQTALEHPEL SELKTVTGTN PVFAGANYAA WAVNVAQVID SETADNLEKT TAALSILPGI GSVMGIADGA VHHNTEEIVA QSIALSSLMV AQAIPLVGEL VDIGFAAYNF VESIINLFQV VHNSYNRPAY SPGHKTQPFL HDGYAVSWNT VEDSIIRTGF QGESGHDIKI TAENTPLPIA GVLLPTIPGK LDVNKSKTHI

SVNGRKIRMR CRAIDGDVTF CRPKSPVYVG NGVHANLHVA FHRSSSEKIH SNEISSDSIG VLGYQKTVDH TKVNSKLSLF FEIKS (SEQ ID NO:1) In one embodiment, CRM197 is isolated from cultures of C7 strain of Corynebacterium diphtheria (197) grown in casamino acids and yeast extract-based medium. In another embodiment, CRM197 is prepared recombinantly according to the methods described in US 5,614,382. Typically, CRM197 is purified through a combination of ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography.

In some embodiments, CRM197 is prepared on Pseudomonas fluorescens using Pfenex Expression Technology™ (Pfenex Inc., San Diego, CA). Other suitable carrier proteins include additional inactivated bacterial toxins such as DT (Diphtheria toxoid) or DT fragment B (DTFB), TT (tetanus toxoid) or TT fragment C, pertussis toxoid, cholera toxoid (e.g. as described in WO 2004 /083251), E. coli LT, E. coli ST, and Pseudomonas aeruginosa exotoxin A.

Bacterial outer membrane proteins such as outer membrane protein complex (OMPC), porins, transferrin binding proteins, pneumococcal surface protein A (PspA; See WO 02/091998), pneumococcal adhesin protein (PsaA), peptidase C5a of Group A or Group B streptococcus, or protein D Haemophilus influenzae, pneumococcal pneumolysin (Kuo et al., 1995, Infect Immun 63; 2706-13) including ply detoxified in some way, for example, dPLY-GMBS (See WO 04 /081515) or dPLY-formaldehyde, PhtX, including PhtA, PhtB, PhtD, PhtE and Pht protein fusions, e.g. PhtDE fusions, PhtBE fusions (See WO 01/98334 and WO 03/54007), can also be used.

Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified tuberculin protein derivative (PPD), PorB (from N. meningitidis), PD (protein D from Haemophilus influenzae; see, for example, EP 0 594 610 B ), or immunologically functional equivalents thereof, synthetic peptides (See EP0378881 and EP0427347), heat shock proteins (See WO 93/17712 and WO 94/03208), pertussis proteins ( See WO 98/58668 and EP0471177), cytokines, lymphokines, growth factors or hormones (See WO 91/01146), artificial proteins comprising multiple human CD4+ T cell epitopes of various pathogen-derived antigens (See Falugi et al., 2001). , Eur J Immunol 31:

3816 to 3824) as N19 protein (See Baraldoi et al., 2004, Infect Immun 72:

4,884 to 4,887), iron uptake proteins (See WO 01/72337), C. difficile toxin A or B (See WO 00/61761), and flagellin (See Ben-Yedidia et al., 1998, Immunol Lett 64 :9) can also be used as carrier proteins. Other DT mutants can be used as the carrier protein, such as CRM176, CRM228, CRM45 (Uchida et al., 1973, J Biol Chem 218:3838-3844); CRM9, CRM45, CRM102, CRM103 and CRM107 and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Editors: Frankel, Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Gly and other mutations disclosed in US 4,709,017 or US 4,950,740; mutation of at least one or more residues Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutations disclosed in US 5,917,017 or US Patent

6,455,673; or fragment disclosed in US 5,843,711. Such DT mutants can also be used to produce DTFB variants wherein the variants comprise the B fragment which contains the epitope regions. In certain embodiments, the carrier protein is selected from the group consisting of: Outer Membrane Protein Complex (OMPC), tetanus toxoid, diphtheria toxoid, protein D, and CRM197. In some embodiments of the present invention, a second carrier may be used for one or more of the polysaccharide-protein conjugates in the multivalent immunogenic composition. The second carrier protein is preferably a protein that is non-toxic and non-reactogenic and obtainable in sufficient quantity and purity. The second carrier protein is also conjugated or linked to the S. pneumoniae polysaccharide to increase the immunogenicity of the antigen. Carrier proteins must be amenable to standard conjugation procedures.

In one embodiment, each capsular polysaccharide not conjugated to the first carrier protein is conjugated to the same second carrier protein (e.g., each capsular polysaccharide molecule that is conjugated to a single carrier protein). In another embodiment, the capsular polysaccharides not conjugated to the first carrier protein are conjugated to two or more carrier proteins (each capsular polysaccharide molecule is conjugated to a single carrier protein). In such embodiments, each capsular polysaccharide of the same serotype is typically conjugated to the same carrier protein.

In embodiments of the present invention, including Embodiment E1 and any submodality thereof, one or more (including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more, where applicable) of the polysaccharide serotypes is conjugated to CRM197. In additional embodiments of the present invention, including Embodiment E1 and any submodality thereof, each of the polysaccharide serotypes is conjugated to CRM197. Formulation of the polysaccharide-protein conjugates of the present invention can be accomplished using methods recognized in the art.

For example, individual pneumococcal conjugates can be formulated with a physiologically acceptable carrier to prepare the composition.

Examples of such carriers include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.

In a preferred embodiment, the vaccine composition is formulated in L-histidine buffer with sodium chloride.

In some embodiments of the present invention, the multivalent immunogenic composition comprises multiple polysaccharide-protein conjugates from S. pneumoniae comprising capsular polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein and an adjuvant, wherein the S. pneumoniae serotypes are as described in the present invention.

Adjuvants suitable for enhancing the effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (defined below) or bacterial cell wall components), such as (a) MF59 (International Patent Application Publication WO 90 /14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as a Model 110Y microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP microfluidized into a submicron emulsion or vortexed to generate a micron-sized emulsion. larger particle, (c) Ribi™ Adjuvant System (RAS), (Corixa, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of 3-O-deacetylated monophosphorylipid A (MPL™) described in US Patent 4,912,094, d trehalose imicolate (TDM), and cell wall structure (CWS), preferably MPL+CWS (Detox™); and (d) a Montanide ISA; (3) saponin adjuvants such as Quil A or STIMULON™ QS-21 (Antigenics, Framingham, MA) (see, for example, US Patent 5,057,540) can be used or particles generated therefrom as ISCOMs immunostimulants formed by the combination of cholesterol, saponin, phospholipid and amphipathic proteins) and Iscomatrix® (which have essentially the same structure as an ISCOM, but without the protein); (4) bacterial lipopolysaccharides, synthetic lipid analogs A such as aminoalkyl glucosamine phosphate (AGP) compounds, or derivatives or analogs thereof, which are available from Corixa, and which are described in US Patent 6,113,918; one such AGP is 2-[( 2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-ß-D-glucopyranoside R)-3-tetradecanoyloxytetradecanoylamino]ethyl, which is also known as 529 (formerly known as RC529), which is formulated as an aqueous form or as a stable emulsion (5) synthetic polynucleotides as oligonucleotides containing CpG motif(s) (Patent US 6,207,646); (6) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.) , interferons (eg, interferon gamma), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, etc.; and (7) complement, as a C3d complement component trimer.

In another embodiment, the adjuvant is a mixture of 2, 3 or more of the above adjuvants, for example, SBAS2 (an oil-in-water emulsion also containing 3-deacetylated monophosphoryl lipid A and QS21). Muramyl peptides include, but are not limited to, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramil-L-alanine-2-(1', 2' dipalmitoyl-sn -glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

In certain embodiments, the adjuvant is an aluminum salt.

The aluminum salt adjuvant may be a precipitated alum vaccine or an alum adsorbed vaccine.

Aluminum salt adjuvants are well known in the art and are described, for example, in Harlow, E. and D.

Lane (1988; Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992; Aluminum salts.

Research in Immunology 143: 489 to 493). Aluminum salt includes, but is not limited to, hydrated alumina, alumina hydrate, alumina trihydrate (ATH), aluminum hydrate, aluminum trihydrate, alhydrogel, Superfos, Amphogel, aluminum(III) hydroxide , aluminum hydroxyphosphate sulfate, Aluminum Phosphate Adjuvant (APA), amorphous alumina, alumina trihydrate or trihydroxyaluminium.

APA is an aqueous suspension of aluminum hydroxyphosphate.

APA is manufactured by mixing aluminum chloride and sodium phosphate in a 1:1 volumetric ratio to precipitate aluminum hydroxyphosphate.

After the blending process, the material is reduced in size with a high shear mixer to obtain a monodisperse particle size distribution.

The product is then diafiltered against physiological saline and sterile steam.

In one embodiment, the dose of aluminum salt is 10, 15, 20, 25, 30, 50, 70, 100, 125, 150, 200, 300, 500, or 700 µg, or 1, 1.2, 1.5 , 2, 3, 5 mg or more.

In yet another embodiment, the dose of alum salt described above is per µg of recombinant protein.

In certain embodiments, a commercially available Al(OH)3 (e.g., Alhydrogel or Superfos from Denmark/Accurate Chemical and Scientific Co., Westbury, NY) is used to adsorb proteins at a rate of 50 to 200 µg protein/mg of aluminum hydroxide.

Protein adsorption is dependent, in another embodiment, on the pI (Isoelectric pH) of the protein and the pH of the medium.

A protein with a lower pI adsorbs to the positively charged aluminum ion more strongly than a protein with a higher pI.

Aluminum salts may establish an antigen depot that is slowly released over a period of 2 to 3 weeks, be involved in nonspecific macrophage and complement activation, and/or stimulate the innate immune mechanism (possibly through uric acid stimulation). See, for example, Lambrecht et al., 2009, Curr Opin Immunol 21:23. Monovalent bulk aqueous conjugates are typically mixed together and diluted to target 4 µg/mL for all serotypes except 6B, which can be diluted to target 8 µg/mL.

Once diluted, the batch will be filter sterilized, and an equal volume of aluminum phosphate adjuvant added aseptically to target a final aluminum concentration of 250 µg/mL.

The formulated and adjuvanted batch will be filled in ampoules of 0.5 mL/dose for single use.

In certain embodiments, the adjuvant is a CpG-containing nucleotide sequence, for example, a CpG-containing oligonucleotide, in particular, a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment, the adjuvant is ODN 1826, obtainable from Coley Pharmaceutical Group.

Methods for using CpG oligonucleotides are well known in the art and are described, for example, in Sur et al., 1999, J Immunol. 162:6284 to 6293; Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al., 2006, Crit Rev Ther Drug Carrier Syst. 23:89-110. In alternative embodiments, the immunogenic composition comprises multiple polysaccharide-S. pneumoniae protein conjugates as described in the present invention, for example, in Embodiment E1 or any submodality thereof, and does not comprise an adjuvant.

Formulations

The multivalent immunogenic compositions of the present invention may be formulated as single-dose ampoules, multi-dose ampoules, or as pre-filled glass or plastic syringes.

In another embodiment, the multivalent immunogenic compositions of the present invention are administered orally, and are thus formulated in a form suitable for oral administration, i.e., as a solid or liquid preparation.

Solid oral formulations include tablets, capsules, pills, granules, pellets and the like.

Liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.

Pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils.

Examples of non-aqueous solvents are propylene glycol, polyethylene glycol and injectable organic esters such as ethyl oleate.

Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.

Examples of oils are those of animal, vegetable or synthetic origin, for example peanut oil, soybean oil, olive oil, sunflower oil, fish liver oil, other marine oil or a milk or egg lipid.

The multivalent immunogenic compositions of the present invention may be isotonic, hypotonic or hypertonic.

However, it is often preferred that a composition for infusion or injection be essentially isotonic when administered.

Thus, for storage, a composition can be preferably isotonic or hypertonic.

If the composition is hypertonic for storage, it may be diluted to an isotonic solution prior to administration.

The isotonic agent can be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate.

Examples of ionic isotonic agents include, but are not limited to, NaCl, CaCl2, KCl and MgCl2. Examples of non-ionic isotonic agents include, but are not limited to, mannitol, sorbitol and glycerol.

It is also preferred that at least one pharmaceutically acceptable additive is a buffer.

For some purposes, for example when the pharmaceutical composition is intended for infusion or injection, it is often desired that the composition comprises a buffer, which has the ability to buffer a solution to a pH in the range of 4 to 10, such as 5 to 9. , for example, 6 to 8. The buffer may, for example, be selected from the group consisting of TRIS buffer, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine.

The buffer may be selected from USP compatible buffers for parenteral use, in particular when the pharmaceutical formulation is for parenteral use.

For example, the buffer can be selected from the group consisting of monobasic acids such as acetic, benzoic, gluconic, glyceric and lactic; dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric, polybasic acids such as citric and phosphoric; and bases such as ammonia, diethanolamine, glycine, triethanolamine and TRIS.

Parenteral vehicles (for subcutaneous, intravenous, intra-arterial or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's oils and fixed.

Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.

Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.

In general, water, saline,

aqueous dextrose and related sugar solutions, glycols such as propylene glycols or polyethylene glycol, Polysorbate 80 (PS-80), Polysorbate 20 (PS-20), and Poloxamer 188 (P188) are preferred liquid carriers, particularly for injectable solutions.

Examples of oils are those of animal, vegetable or synthetic origin, for example peanut oil, soybean oil, olive oil, sunflower oil, fish liver oil, other marine oil or a milk or egg lipid.

The formulations of the present invention may also contain a surfactant.

Preferred surfactants include, but are not limited to: Poloxamer-188 (P188; Pluronic; F68 NF), polyoxyethylene sorbitan ester surfactants (commonly called Tweens), especially PS-20 and PS-80; copolymers of ethylene oxide (EO), propylene oxide (PO) and/or butylene oxide (BO), sold under the brand name DOWFAX™, as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1,2-ethanedi-yl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); ethoxylated non-phenols such as Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as bBrij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate.

A preferred surfactant for inclusion in the emulsion is PS-80. Surfactant blends can be used, for example PS-80/Span 85 blends. The combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (PS-80) and an octoxynol such as t-octylphenoxypolyethoxyethanol ( Triton X-100) is also suitable.

Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylene sorbitan esters (such as PS-80) from 0.01 to 1%, in particular about 0.1%; octyl or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) from 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) from 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%. In certain embodiments, the composition consists essentially of histidine (20 mM), saline (150 mM), and 0.2% PS-20 at a pH of 5.8 with 250 µg/mL APA (Aluminum Phosphate Adjuvant) . PS-20 can be in the range of 0.005% to 0.3% (w/v). In another embodiment, PS-20 may be in the range of 0.025% to 0.8% (w/v). In another embodiment, PS-20 may be in the range of 0.05% to 0.8% (w/v). In another embodiment, PS-20 may be in the range of 0.05% to 0.2% (w/v). The process consists of combining a blend of up to 24 serotypes in histidine, saline and PS-20, then combining this blended material with APA and saline with or without antimicrobial preservatives.

In particular embodiments, the multivalent immunogenic composition comprises S. pneumoniae protein-polysaccharide conjugates wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes in the conjugates of polysaccharide-protein comprise any of the serotype sets set forth in the present invention, and additionally comprise 20 to 80 mM histidine pH 5.8 and 150 mM NaCl.

In some embodiments, the multivalent immunogenic composition additionally comprises from 0.2% to 0.8% w/v polysorbate 20.

The PCV24 multivalent immunogenic composition is prepared by individually conjugating the CRM197 protein to polysaccharide (PnPs) from S. pneumoniae serotypes -1, -3, -4, -5, -6A, -6B, -7F, -8, - 9V, -10A, -11A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F and -35B using reductive amination in an aprotic solvent (also referred to as DMSO chemical) and formulated in 20 mM L-Histidine pH 5.8, 150 mM NaCl and 0.1 % w/v Polysorbate-20 (PS-20) at 4 µg/mL or 8 µg/mL of each polysaccharide serotype for a total polysaccharide concentration of 96 µg/mL or 192 µg/mL, respectively, and called “PCV24 without adj”. In another specific embodiment, the PCV24 multivalent immunogenic composition is prepared in 20 mM L-Histidine pH 5.8, 150 mM NaCl and 0.2 % w/v Polysorbate-20 (PS-20) at 4 µg/mL of each polysaccharide serotype to a total polysaccharide concentration of 96 µg/ml further comprising 250 µg [Al]/ml in the form of Aluminum Phosphate Adjuvant.

This is called “PCV24/APA”. The choice of surfactant may need to be optimized for different drug products and drug substances.

For multivalent vaccines that have 15 or more serotypes, PS-20 and P188 are preferred.

The choice of chemical used to prepare the conjugates can also play an important role in stabilizing the formulation.

In particular, when the conjugation reactions used to prepare different polysaccharide-protein conjugates in a multivalent composition include both aqueous solvent and DMSO solvent, particular surfactant systems provide significant differences in stability.

Improved stability of polysaccharide-protein conjugates was observed with polysorbate 20 alone or with poloxamer 188 in combination with a polyol.

The exact mechanism of how a specific detergent protects a biotherapy is insufficiently understood and cannot be predicted a priori. Possible stabilization mechanisms include preferential hydration, preferential exclusion, air/Liquid interface competition between the biotherapy and the surface, surface tension and/or direct association of the detergent with the biotherapy to mask hydrophobic adhesives that serve as seeds for aggregation. Poloxamer can also be used in the compositions of the present invention. A poloxamer is a nonionic triblock copolymer composed of a central hydrophobic polyoxypropylene (poly(propylene oxide)) chain flanked by two hydrophilic polyoxyethylene (poly(ethylene oxide)) chains. Poloxamers are also known by the brand name Pluronic®. Because polymer block lengths can be customized, there are many different poloxamers that have slightly different properties. For the generic term "poloxamer", these copolymers are commonly named by the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 give the proper molecular weight of the polyoxypropylene core, and the last digit x 10 gives the percentage of polyoxyethylene content (eg P407 = Poloxamer with a polyoxypropylene molecular weight of 4000 g/mol and a polyoxyethylene content of 70 %). For the brand name Pluronic®, the coding of these copolymers begins with a letter to define their physical form at room temperature (L = liquid, P = paste, F = flake (solid)) followed by two or three digits. The first digit (two digits in a three-digit number) in the numerical designation, multiplied by 300, indicates the approximate molecular weight of the hydrophobe; and the last digit x 10 gives the percentage of polyoxyethylene content (e.g. L61 = Pluronic® with a polyoxypropylene molecular weight of

1,800 g/mol and a polyoxyethylene content of 10 %). See US Patent 3,740,421.

Examples of poloxamers have the general formula: HO(C2H4O)a(C3H6O)b(C2H4O)aH, where blocks a and b have the following values: Pluronic® Poloxamer AB Molecular weight L31 2 16 1,100 (average) L35 1,900 (average) L44NF 124 12 20 2.090 to 2.360 L64 2.900 (mean) L81 2,800 (mean) L121 4,400 (mean) P123 20 70 5.750 (mean) F68NF 188 80 27 7.680 to 9.510 F87NF 237 64 37 6.840 to 8.830 F108nf 338 141 44 12,700 to 17,400 F127NF 407 101 56 9840 to 14600 Molecular weight units as used in the present invention are in Dalton (Da) or g/mol. Preferably, the poloxamer generally has a molecular weight in the range of 1,100 to 17,400 Da, 7,500 to 15,000 Da, or 7,500 to

10,000 Da. Poloxamer can be selected from poloxamer 188 or poloxamer 407. The final concentration of poloxamer in the formulations is

0.001% to 5% weight/volume, or 0.025% to 1% weight/volume. In certain aspects, the polyol is propylene glycol and is in the final concentration of 1% to 20% weight/volume. In certain aspects, the polyol is polyethylene glycol 400 and is in a final concentration of 1% to 20% weight/volume. Suitable polyols for the formulations of the present invention are polymeric polyols, particularly polyether diols which include, but are not limited to, propylene glycol and polyethylene glycol, polyethylene glycol monomethyl ethers. Propylene glycol is available in a monomer molecular weight range of ~425 to ~2,700. Polyethylene Glycol and Polyethylene Glycol Monomethyl Ether is also available in a molecular weight range ranging from ~200 to ~35,000 including, but not limited to, PEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600, PEG MME 2000, PEG MME 3350 and PEG MME 4000. A preferred polyethylene glycol is polyethylene glycol

400. The final concentration of the polyol in the formulations of the present invention can be from 1% to 20% weight/volume or 6% to 20% weight/volume. The formulation also contains a pH buffered saline solution. The buffer may, for example, be selected from the group consisting of TRIS buffer, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, HEPES (4-(acid) 2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) and triethanolamine. The buffer has the ability to buffer a solution to a pH in the range of 4 to 10, 5.2 to 7.5, or 5.8 to 7.0. In one aspect of the present invention, the buffer is selected from the group consisting of phosphate, succinate, histidine, MES, MOPS, HEPES, acetate or citrate. The buffer may additionally, for example, be selected from USP compatible buffers for parenteral use, in particular when the pharmaceutical formulation is for parenteral use. In one embodiment, the concentration of buffer will be in the range of 1 mM to 100 mM. In another embodiment, the buffer concentration will be in the range of 10 mM to 80 mM. In another embodiment, the buffer concentration will be in the range of 1 mM to 50 mM, or 5 mM to 50 mM. In certain aspects, the buffer is histidine at a final concentration of 5 mM to 50 mM, or succinate at a final concentration of 1 mM to 10 mM. In certain aspects, the histidine buffer is at a final concentration of 20 mM ± 2 mM. While saline (for example, a solution containing NaCl)

is preferred, other salts suitable for formulation include, but are not limited to, CaCl 2 , KCl and MgCl 2 and combinations thereof.

Non-ionic isotonic agents including, but not limited to, sucrose, trehalose, mannitol, sorbitol and glycerol may be used in place of a salt.

Suitable salt ranges include, but are not limited to, 20 mM to 500 mM or 40 mM to 170 mM.

In one aspect, the saline is NaCl, optionally present at a concentration of 25 mM to 170 mM.

In a preferred embodiment, the formulations comprise a buffer of L-histidine with sodium chloride.

In another embodiment, the pharmaceutical composition is delivered in a controlled-release system.

For example, the agent can be administered using intravenous infusion, a transdermal patch, liposomes, or other modes of administration.

In another embodiment, polymeric materials are used; for example, in microspheres in or an implant.

The amount of conjugate in each dose of the composition is selected as an amount that induces an immunoprotective response without significant adverse effects.

Such amount may vary depending on the pneumococcal serotype.

Generally, for polysaccharide-based conjugates, each dose will comprise 0.08 to 100 µg of each polysaccharide.

In some embodiments of the present invention, the dose of each polysaccharide conjugate is from 0.08 to 10 µg.

In additional embodiments, the dose of each conjugate is 1 to 5 µg, 0.4 to 4 µg, 0.4 to 3 µg, 0.4 to 2 µg, or 0.4 to 2 µg. 1 g.

In some embodiments, the dose of one or more polysaccharide conjugates is 100, 150, 200, 250, 300, 400, 500, or 750 ng or 0.4, 0.5, 0.6, 0.7, 0 .75, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90, or 100 µg.

In some embodiments of the compositions of the present invention,

all of the polysaccharide conjugates are present in the composition in the same amount.

In additional embodiments, the polysaccharide conjugates are present in the composition in different amounts (i.e., at least one polysaccharide conjugate is present in an amount that is different from one or more of the other polysaccharide conjugates in the composition). The optimal amounts of components for a particular immunogenic composition can be determined by standard studies that involve observation of adequate immune responses in subjects.

For example, in another embodiment, the dosage for human vaccination is determined by extrapolation from animal studies to human data.

In another embodiment, the dosage is determined empirically.

The compositions of this present invention may also include one or more S. pneumoniae proteins.

Examples of S. pneumoniae proteins suitable for inclusion include those identified in International Patent Application Publications WO 02/083855 and WO 02/053761. In certain embodiments, the compositions of the present invention are administered to a subject by one or more methods known to one skilled in the art, such as parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, intranasally, subcutaneously, intraperitoneally and formulated accordingly.

In one embodiment, the compositions of the present invention are administered via epidermal injection, intramuscular injection, intravenous, intraarterial, subcutaneous, or intrarespiratory mucosal injection of a liquid preparation.

Liquid formulations for injection include solutions and the like.

III.

Preparation Methods

Streptococcus pneumoniae capsular polysaccharides can be prepared by standard techniques known to those skilled in the art.

For example, polysaccharides can be isolated from bacteria and can be sized to some degree by known methods (see, for example, European Patents EP497524 and EP497525); and preferably by microfluidization obtained using a homogenizer or by chemical hydrolysis.

In one embodiment, each pneumococcal polysaccharide serotype is cultured in a soy-based medium.

The individual polysaccharides are then purified through standard steps that include centrifugation, precipitation and ultrafiltration.

See, for example, US Patent Application Publication 2008/0286838 and US Patent 5,847,112. Polysaccharides can be sized to reduce viscosity in polysaccharide samples and/or to improve filterability for conjugated products using techniques such as mechanical or chemical sizing.

Chemical hydrolysis can be conducted using acetic acid.

Mechanical sizing can be conducted using High Pressure Homogenizing Shear.

Purified polysaccharides can be chemically activated to prepare saccharides capable of reacting with the carrier protein.

Purified polysaccharides can be attached to a ligand.

Once activated or linked to a ligand, each capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate.

Polysaccharide conjugates can be prepared by known coupling techniques.

The polysaccharide can be coupled to a linker to form a polysaccharide linker intermediate in which the free terminus of the linker is an ester group.

The linker is therefore one in which at least one terminus is an ester group.

The other terminus is selected so that it can react with the polysaccharide to form the polysaccharide linker intermediate.

The polysaccharide can be coupled to a linker using a primary amine group on the polysaccharide.

In this case, the linker typically has an ester group at both ends.

This allows coupling to occur by reacting one of the ester groups with the primary amine group on the polysaccharide by nucleophilic acyl substitution.

The reaction results in a polysaccharide linker intermediate in which the polysaccharide is coupled to the linker via an amide bond.

The linker is therefore a bifunctional linker that provides a first ester group to react with the primary amine group on the polysaccharide and a second ester group to react with the primary amine group on the carrier molecule.

A typical ligand is adipic acid N-hydroxysuccinimide diester (SIDEA). Coupling can also occur indirectly, that is, with an additional linker that is used to derive the polysaccharide before coupling to the linker.

The polysaccharide is coupled to the additional linker using a carbonyl group at the reducing end of the polysaccharide.

This coupling comprises two steps: (a1) reacting the carbonyl group with the additional ligand; and (a2) reacting the free end of the additional linker with the linker.

In these embodiments, the additional linker typically has a primary amine group at both ends, therefore allowing step (a1) to occur by reacting one of the primary amine groups with the carbonyl group on the polysaccharide by reductive amination.

A primary amine group is used, which is reactive with the carbonyl group on the polysaccharide.

Hydrazide or hydroxylamino groups are suitable.

The same primary amine group is typically present at both ends of the additional linker.

The reaction results in an additional polysaccharide linker intermediate in which the polysaccharide is coupled to the additional linker via a C—N bond. The polysaccharide can be coupled to the additional linker using a different group on the polysaccharide, particularly a carboxyl group. This coupling comprises two steps: (a1) reacting the group with the additional linker; and (a2) reacting the free end of the additional linker with the linker. In this case, the additional linker typically has a primary amine group at both ends, therefore allowing step (a1) to occur by reacting one of the primary amine groups with the carboxyl group on the polysaccharide by activating EDAC. A primary amine group is used, which is reactive with the EDAC-activated carboxyl group on the polysaccharide. A hydrazide group is suitable. The same primary amine group is typically present at both ends of the additional linker. The reaction results in an additional polysaccharide linker intermediate in which the polysaccharide is coupled to the additional linker via an amide bond. In one embodiment, chemical activation of the polysaccharides and subsequent conjugation to the carrier protein by reductive amination can be accomplished by means described in US Patents 4,365,170, 4,673,574 and

4,902,506 , US Patent Application Publications 2006/0228380 , 2007/184072 , 2007/0231340 and 2007/0184071 , and WO2006/110381 , WO2008/079653 and WO2008/143709 . The chemical may include activating pneumococcal polysaccharide by reaction with any oxidizing agent that oxidizes a terminal hydroxyl group to an aldehyde, such as periodate (including sodium periodate, potassium periodate, or periodic acid). The reaction leads to a random oxidative cleavage of vicinal hydroxyl groups of carbohydrates with the formation of reactive aldehyde groups. Coupling to the carrier protein is by reductive amination through direct amination to the lysyl groups of the protein. For example, conjugation can be accomplished by reacting a mixture of the activated polysaccharide and carrier protein with a reducing agent such as sodium cyanoborohydride.

The conjugation reaction can take place under aqueous solution or in the presence of DMSO.

See, for example, US 2015/0231270 , US 2011/0195086 and EP 0471 177 B1 . The unreacted aldehydes are then capped with the addition of a strong reducing agent such as sodium borohydride.

Reductive amination involves two steps, (1) oxidation of the polysaccharide to form reactive aldehydes, (2) reduction of the imine (Schiff base) formed between activated polysaccharide and a carrier protein to form a stable amine conjugate bond.

Prior to oxidation, the polysaccharide is optionally reduced in size.

Mechanical methods (eg homogenization) or chemical hydrolysis may be employed.

Chemical hydrolysis can be conducted using acetic acid.

The oxidation step may involve reaction with periodate.

For purposes of the present invention, the term "periodate" includes both periodate and periodic acid; the term also includes both metaperiodate (104-) and orthoperiodate (106-) and includes the various periodate salts (eg, sodium periodate and potassium periodate). In one embodiment, the capsular polysaccharide is oxidized in the presence of metaperiodate, preferably in the presence of sodium periodate (NaIO4 ). In another embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably in the presence of periodic acid.

In one embodiment, the oxidizing agent is a radical-stable nitroxyl compound or nitroxide, such as piperidine-N-oxy or pyrrolidine-N-oxy compounds, in the presence of an oxidant to selectively oxidize primary hydroxyls (as described in WO 2014/097099) . In said reaction, the real oxidant is the N-oxoammonium salt, in a catalytic cycle.

In one aspect, said radical-stable nitroxyl compound or nitroxide is piperidine-N-oxy or pyrrolidine-N-oxy compounds.

In one aspect, said stable nitroxyl radical compound or nitroxide bears a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In one aspect, said stable nitroxyl radical compound is TEMPO or a derivative thereof.

In one aspect, said oxidant is a molecule bearing an N-halo moiety.

In one aspect, said oxidant is selected from the group consisting of N-Chlorosuccinimide, N-Bromosuccinimide, N-lodosuccinimide, Dichloroisocyanuric acid, 1,3,5-Trichloro-1,3,5-triazinane-2,4 ,6-Trione, Dibromoisocyanuric Acid, 1,3,5-Tribromo-1,3,5-triazinane-2,4,6-Trione, Diiodoisocyanuric Acid and 1,3,5-Triiodo-1,3 ,5-triazine-2,4,6-trione.

Preferably, said oxidant is N-Chlorosuccinimide.

In certain aspects, the oxidizing agent is 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) free radical and N-Chlorosuccinimide (NCS) as the co-oxidant (as described in WO 2014/097099). Therefore, in one aspect, S. pneumoniae glycoconjugates are obtainable by a method comprising the steps of: a) reacting a saccharide with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to produce an activated saccharide; and b) reacting the activated saccharide with a carrier protein comprising one or more amine groups (said method is hereinafter referred to as "reductive amination of TEMPO/NCS"). Optionally, the oxidation reaction is cooled by adding a cooling agent.

The cooling agent can be selected from vicinal diols, 1,2-aminoalcohols, amino acids, glutathione, sulfite, bisulfate, dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites or phosphorous acid (such as glycerol, ethylene glycol, propan-1,2 -diol, butan-1,2-diol or butan-2,3-diol, ascorbic acid). In certain embodiments, the present invention provides a method for preparing a serotype 8 Streptococcus pneumoniae polysaccharide-protein conjugate using a conjugation reaction in an aprotic solvent, wherein the conjugation reaction does not use cyanoborohydride.

In additional embodiments, the conjugation reaction is a base-Schiff reduction or reductive amination.

In additional embodiments, the protein is tetanus toxoid, diphtheria toxoid, or CRM197. In still further embodiments, the protein is CRM197. In additional embodiments, the conjugation reaction is reductive amination.

In additional embodiments, the reductive amination is performed on dimethylsulfoxide (DMSO). In some embodiments, the polysaccharides oxidized prior to conjugation have a molecular weight between 30 kDa and 1000 kDa.

Molecular weight can be calculated by size exclusion chromatography (SEC) combined with multi-angle light scattering detector (MALS) and refractory index (RI) detector. In some embodiments, the polysaccharide has a molecular weight between 50 kDa and 300 kDa.

In some embodiments, the polysaccharide has a molecular weight between 50 kDa and 1000 kDa.

In additional embodiments, the polysaccharide has a molecular weight between 70 kDa and 900 kDa.

In other embodiments, the polysaccharide has a molecular weight between 100 kDa and 800 kDa.

In other embodiments, the polysaccharide has a molecular weight between 200 kDa and 600 kDa.

In additional embodiments, the polysaccharide has a molecular weight of 100 kDa to 1000 kDa; 100 kDa to 900 kDa; 100 kDa to 800 kDa; 100 kDa to 700 kDa; 100 kDa to 600 kDa; 100 kDa to 500 kDa; 100 kDa to 400 kDa; 100 kDa to 300 kDa; 150 kDa to 1000 kDa; 150 kDa to 900 kDa; 150 kDa to 800 kDa; 150 kDa to 700 kDa; 150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 150 kDa to 300 kDa; 200 kDa to 1000 kDa; 200 kDa to 900 kDa; 200 kDa to 800 kDa; 200 kDa to 700 kDa; 200 kDa to 600 kDa; 200 kDa to 500 kDa; 200 kDa to 400 kDa; 200 kDa at 300; 250 kDa to 1000 kDa; 250 kDa to 900 kDa; 250 kDa to 800 kDa; 250 kDa to 700 kDa;

250 kDa to 600 kDa; 250 kDa to 500 kDa; 250 kDa to 400 kDa; 250 kDa to 350 kDa; 300 kDa to 1000 kDa; 300 kDa to 900 kDa; 300 kDa to 800 kDa; 300 kDa to 700 kDa; 300 kDa to 600 kDa; 300 kDa to 500 kDa; 300 kDa to 400 kDa; 400 kDa to 1000 kDa; 400 kDa to 900 kDa; 400 kDa to 800 kDa; 400 kDa to 700 kDa; 400 kDa to 600 kDa; or 500 kDa to 600 kDa.

The second step of the conjugation process is the reduction of the imine bond (Schiff base) between activated polysaccharide and a carrier protein to form a stable conjugate bond (so-called reductive amination) using a reducing agent.

Reducing agents that are suitable include cyanoborohydrides (such as sodium cyanoborohydride or sodium borohydride). In one embodiment, the reducing agent is sodium cyanoborohydride.

In certain embodiments, the reductive amination reaction is carried out in an aprotic solvent (or a mixture of aprotic solvents). In one embodiment, the reduction reaction is carried out in DMSO solvent or in DMF (dimethylformamide) solvent. DMSO or DMF solvent can be used to reconstitute activated polysaccharide and carrier protein, if lyophilized.

In one embodiment, the aprotic solvent is DMSO.

At the end of the reduction reaction, there may be unreacted aldehyde groups remaining in the conjugates, which can be capped using a suitable capping agent.

In one embodiment, such capping agent is sodium borohydride (NaBH4). Suitable alternatives include sodium or sodium triacetoxyborohydride or zinc borohydride in the presence of Bronsted or Lewis acids), amine boranes such as pyridine boranes, 2-Picolino borane, 2,6-diborane methanol, dimethylamine -borane, t-BuMe'PrN-BH3, benzylamine-BH3 or 5-ethyl-2-methylpyridine (PEMB) boranes or borohydride exchange resin.

After conjugation (the reduction reaction and optionally capping), the glycoconjugates can be purified (enriched with respect to the amount of polysaccharide-protein conjugate) by a variety of techniques known to those skilled in the art.

These techniques include dialysis, concentration/diafiltration operations, tangential flow filtration, precipitation/elution, column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE or hydrophobic interaction chromatography) and deep filtration.

In one embodiment, the glycoconjugates are purified by diafiltration or ion exchange chromatography or size exclusion chromatography.

Glycoconjugates prepared using reductive amination in an aprotic solvent are generally used in multivalent pneumococcal conjugate vaccines.

Thus, in certain embodiments for multivalent compositions where not all serotypes are prepared in an aprotic solvent, the reduction reaction for the remainder of the serotypes is carried out in an aqueous solvent (e.g. selected from PBS (phosphate-buffered saline). ), MES (2-(N-morpholino)ethanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Bis-Tris, ADA (N-(2-Acetamido)iminodiacetic acid), PIPES ( piperazine-N,N′-bis(2-ethanesulfonic acid)), MOPSO (3-Morpholino-2-hydroxypropanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS ( 3-(N-morpholino)propanesulfonic acid), DIPSO (3-Bis(2-hydroxyethyl)amino-2-hydroxypropane-1-sulfonic acid), MOBS (4-(N-morpholino)butanesulfonic acid), HEPPSO (N-( 2-Hydroxyethyl)piperazine-N׳-(2-hydroxypropanesulfonic acid)), POPSO (Piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA (triethanolamine), EPPS (4- (2-Hydroxyethyl)piperazine-1-propa sulfonic acid), or Bicin at a pH between 6.0 and 8.5, 7.0 and 8.0, or 7.0 and 7.5). S. pneumoniae protein-polysaccharide capsular conjugates that can be prepared using reductive amination in an aprotic solvent include, but are not limited to, S. pneumonia and serotypes: 1, 3, 4, 5, 6A, 6B, 6C , 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

Polysaccharides can be used in the form of oligosaccharides.

These are conveniently formed by fragmentation of purified polysaccharide (eg, by hydrolysis), which will often be followed by purification of fragments of the desired size.

In certain embodiments, pneumococcal polysaccharide-protein conjugates of one or more of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B are prepared using reductive amination in an aprotic solvent.

In certain embodiments, each of the conjugates in the multivalent immunogenic composition is prepared using reductive amination in an aprotic solvent.

In certain embodiments, polysaccharides of one or more serotypes in a multivalent composition of the present invention are conjugated to a carrier protein using reductive amination in an aprotic solvent and polysaccharides of one or more serotypes are conjugated using reductive amination in an aqueous solvent.

In certain embodiments, polysaccharides of two or more serotypes in a multivalent composition of the present invention are conjugated to a carrier protein using reductive amination in an aprotic solvent.

In other embodiments, polysaccharides of three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty-one or more, twenty-two or more, twenty-three or more, or twenty-four or more serotypes in a multivalent composition of the present invention are conjugated to a carrier protein using reductive amination in an aprotic solvent.

In certain embodiments, polysaccharides of one or more serotypes in a multivalent composition of the present invention are conjugated to a carrier protein using other chemicals which may be in an aprotic solvent or in an aqueous solvent.

Accordingly, the present invention relates to a multivalent immunogenic composition comprising multiple polysaccharide-protein conjugates from S. pneumoniae, each comprising capsular polysaccharide from a serotype of S. pneumoniae conjugated to a carrier protein, wherein the S. pneumoniae are as described in the present invention (i.e., in Section II, “Multivalent Immunogenic Compositions”), wherein the conjugation reaction by which the S. pneumonia polysaccharide of one or more of the polysaccharide-protein conjugates is conjugated to the protein carrier is in an aprotic solvent.

In certain embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of pneumococcal serotypes in a multivalent immunogenic composition are conjugated in an aprotic solvent.

The remaining serotypes are conjugated using an alternative chemical and/or in an aqueous solvent.

It has been determined that the use of DMSO as a solvent during the reductive amination of polysaccharide-protein conjugates results in unexpectedly superior stability and improved immunogenicity for those serotypes relative to the same conjugates prepared under aqueous conditions (See US Serial Application 62/463,216 and 62/555,444). In certain embodiments of the present invention, the total polysaccharide concentration in the composition is from about 0.02 to about 0.288 mg/mL.

In certain embodiments of the present invention, the total polysaccharide concentration in the composition is from about 0.03 to about 0.192 mg/mL.

In certain embodiments of the present invention, the total polysaccharide concentration in the composition is from about 0.04 to about 0.192 mg/mL.

In other embodiments, the total polysaccharide concentration in the composition is from about 0.065 to about 0.096 mg/mL, about 0.070 to about 0.080 mg/mL, about 0.065 to about 0.080 mg/mL, about 0.070 to about 0.070 to about 0.080 mg/mL. about 0.085 mg/mL, about 0.110 to about 0.128 mg/mL, about 0.110 to about 0.175 mg/mL, about 0.10 to about 0.175 mg/mL, about 0.110 to about 0.170 mg /mL, about 0.115 to about 0.15 mg/mL, about 0.110 to about 0.15 mg/mL, about 0.110 to about 0.125 mg/mL, about 0.150 to about 0.170 mg/mL , about 0.150 to about 0.165 mg/mL, about 0.140 to about 0.170 mg/mL, about 0.130 to about 0.170 mg/mL, about 0.150 to about 0.175 mg/mL, about 0.070 to about from 0.170 mg/mL, about 0.065 to about 0.175 mg/mL, or about 0.065 to about 0.180 mg/mL.

In embodiments of the present invention where one or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic compositions are prepared in an aprotic solvent, the total polysaccharide concentration in the composition is stable for 4 weeks or more at 37°C. , 4 weeks or more at 25 °C, or 12 weeks or more at 4 °C.

In certain embodiments of the present invention where one or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic compositions are prepared in an aprotic solvent, the average molecular weight (Mw) of all of the polysaccharide-protein conjugates of S. pneumoniae in composition (average of all conjugates in the composition) is from about 2000 to about 6500 kDa, from about 2500 to about 6000 kDa, from about 3000 to about 5500 kDa, from about 3500 to about 3500 to from about 5,000 kDa, from about 3,500 to about 4,500 kDa, from about 3,500 to about 4,700 kDa, from about 3,500 to about 4,600 kDa, from about 3,500 to about 4,500 kDa, from about 3,500 to about 3,500 from about 4400 kDa, from about 3500 to about 4300 kDa, from about 3500 to about 4200 kDa, from about 3600 to about 4700 kDa, from about 3600 to about 4600 kDa, from about 3600 to about 3600 about 4500 kDa, from about 3600 to about 4400 kDa, from about 3600 to about 4,300 kDa, from about 3,600 to about 4,200 kDa, from about 3,700 to about 4,700 kDa, from about 3,700 to about 4,600 kDa, from about 3,700 to about 4,500 kDa, from about 3,700 to about 3,700 to about 4,600 4400 kDa, from about 3700 to about 4300 kDa, from about 3700 to about 4200 kDa, from about 3800 to about 4700 kDa, from about 3800 to about 4600 kDa, from about 3800 to about 3800 to about 4,600 4,500 kDa, from about 3,800 to about 4,400 kDa, from about 3,800 to about 4,300 kDa, from about 3,800 to about 4,200 kDa, from about 3,900 to about 4,700 kDa, from about 3,900 to about 3,900 to about 4,200 4600 kDa, from about 3900 to about 4500 kDa, from about 3900 to about 4400 kDa, from about 3900 to about 4300 kDa, or from about 3900 to about 4200 kDa. In certain embodiments of the present invention where the polysaccharide-protein conjugates in the multivalent immunogenic compositions are prepared in an aprotic solvent, the Mw of each of the polysaccharide-protein conjugates of S. pneumoniae in the composition (for a single serotype) is of about 1,000 to about 10,000 kDa, from about

1500 to about 5500 kDa, from about 1500 to about 5600 kDa, from about 1500 to about 5600 kDa

1500 to about 5700 kDa, from about 1500 to about 5800 kDa, from about 1500 to about 5800 kDa

1500 to about 5900 kDa, from about 1500 to about 6000 kDa, from about 1500 to about 6000 kDa

1000 to about 5500 kDa, from about 1000 to about 5000 kDa, from about 1000 to about 5000 kDa

1000 to about 4000 kDa, from about 1000 to about 4500 kDa, from about 1000 to about 4500 kDa

1000 to about 4000 kDa, or from about 1000 to about 3500 kDa. In other embodiments, the Mw of a single serotype conjugate in the composition is about 1000 kDa, about 1100 kDa, about 1200 kDa, about 1300 kDa, about 1400 kDa, about 1500 kDa, about 1600 kDa, about 1700 kDa, about 1800 kDa, about 1900 kDa, about 2000 kDa, about 2100 kDa, about 2200 kDa, about 2300 kDa, about 2400 kDa, about 2500 kDa, about 2600 kDa, about 2700 kDa, about 2800 kDa, about 2900 kDa, about 3000 kDa, about 3100 kDa, about 3200 kDa, about 3300 kDa, about 3400 kDa, about 3500 kDa, about 3600 kDa, about 3,700 kDa, about 3,800 kDa, about 3,900 kDa, about 4,000 kDa, about 4,100 kDa, about 4,200 kDa, about 4,300 kDa, about 4400 kDa, about 4,500 kDa, about 4,600 kDa, about 4,700 kDa, about 4,800 kDa, about 4,900 kDa, about 5,000 kDa, about 5,100 kDa, about 5,200 kDa, about 5,300 kDa, about 5,400 kDa, or about 5. 500 kDa. In certain embodiments of the present invention, the polysaccharide-protein conjugates in the multivalent immunogenic compositions are prepared in an aprotic solvent. In certain embodiments, the percentage (as calculated by the number of polysaccharide serotypes prepared in an aprotic solvent divided by the total number of polysaccharide serotypes, where the total number includes those prepared in an aprotic solvent or a protic solvent) of specific conjugates of serotype of S. pneumoniae prepared in an aprotic solvent may be greater than 50%, or greater than 60%, or greater than 70%, or greater than 80%, or greater than 90%, or are 100%. In certain embodiments of the present invention, the serotype 3 protein-polysaccharide conjugate in the composition is prepared in an aprotic solvent and the Mw of said conjugate is from about 1000 to about

5,000 kDa, or from about 1,000 to about 4,000 kDa, or from about 1,000 to about 3,000 kDa, or from about 1,000 to about 2,500 kDa, or from about 1,000 to about 2,500 kDa

1000 to about 2000 kDa.

In certain embodiments of the present invention where one or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic compositions are prepared in an aprotic solvent, the number average molecular weight (Mn) of the polysaccharide-protein conjugates of S. pneumoniae in the composition (average of all conjugates in the composition) is from about 900 to about 3000 kDa, from about

1000 to about 3000 kDa, from about 1000 to about 2500 kDa, from about 1000 to about 2500 kDa

1500 to about 2500 kDa, from about 1800 to about 2500 kDa, from about 1,800 to about 2,500 kDa

1900 to about 2500 kDa, or from about 2000 to about 2500 kDa. In certain embodiments of the present invention where one or more or all of the polysaccharide-protein conjugates in the multivalent immunogenic compositions are prepared in an aprotic solvent, the Mn of each of the polysaccharide-protein conjugates of S. pneumoniae in the composition (for a single serotype) is from about 700 to about 7000 kDa, from about 1000 to about 6000 kDa, from about 1000 to about 5000 kDa, from about 1000 to about 4000 kDa, from about 1000 to about 4000 kDa 1000 to about 3000 kDa, from about 900 to about 5,500 kDa, from about 900 to about 900

5,000 kDa, from about 900 to about 4,500 kDa, from about 900 to about 4,000 kDa, from about 900 to about 3,500 kDa, or from about 900 to about 3,000 kDa. In embodiments of the present invention, the Mw and/or Mn of the polysaccharide-protein conjugates of S, pneumoniae in the composition is stable for 4 weeks or more at 37°C, 4 weeks or more at 25°C, and/or 12 weeks. or more at 4°C. In embodiments of the present invention, the concentration of polysaccharide, Mw, and/or Mn are determined using HPSEC UV/MALS/RI. In some embodiments of the present invention, wherein one or more,

or all of the polysaccharide-protein conjugates in the multivalent immunogenic compositions are prepared in an aprotic solvent, the maximum emission from the composition measured using intrinsic protein fluorescence spectroscopy with an excitation wavelength at 280 nanometers (nm) is about 335 nm to about 342 nm.

In some embodiments, the emission maximum remains from about 335 nm to about 342 nm and the fluorescence intensity is stable for at least 1 week at 37 °C.

In some embodiments, the emission maximum remains from about 335 nm to about 342 nm and the fluorescence intensity is stable for 1 week at 37 °C.

In some embodiments, all of the pneumococcal polysaccharide conjugates in the multivalent composition are prepared using reductive amination in DMSO.

In certain submodalities, the multivalent composition comprising polysaccharide conjugates that have all been prepared using DMSO does not comprise an adjuvant.

Without being bound by any particular theory, a possible mechanism for the improved immunogenicity seen with glycoconjugates prepared in DMSO includes an increased number of bonds between the carbohydrate (capsular polysaccharide) and lysine residues on the surface of the carrier protein that would result in additional attachment points. between the protein and the polysaccharide to impart stability and counteract chemical depolymerization or peptide carbohydrate bond breakage.

See, for example, Hsieh, Characterization of Saccharide-CRM197 Conjugate Vaccines in Brown F, Corbel M, Griffiths E (editors): Physico-Chemical Procedures for the Characterization of Vaccines.

Dev.

Biol.

Basel, Karger, 2000, volume 103, pages 93 to 104. An additional benefit of the increased polysaccharide-protein bonds that are created during conjugation in the DMSO solvent may be additional opportunities for successful peptide-carbohydrate presentation to T cells .

A possible mechanism of improved immunogenicity observed by conjugation in the DMSO solvent may be due to the denaturation of CRM197 in organic solvent, which exposes additional lysines for polysaccharide linkages providing increased chances for glycopeptide presentation on the surface of an APC for T cell-dependent response. for different peptide epitopes.

See Avci et al., 2011, Nature Medicine 17: 1602-1610. Yet another benefit of conjugation in an organic solvent that generates denatured CRM197 in the conjugates may be reduced immunological interference from antibodies against native CRM197 epitopes.

An additional benefit of the increased polysaccharide-protein bonds that are created during conjugation in the DMSO solvent may be the formation of larger-sized polysaccharide-protein conjugates resulting in improved immunogenicity.

The compositions of the present invention are believed to provide significant advantages in eliciting a human response.

In certain embodiments, the conjugation reaction is performed by reductive amination in which nickel is used to improve the efficiency of the conjugation reaction and to aid in the removal of free cyanide.

Transition metals are known to form stable complexes with cyanide and are known to enhance reductive methylation of amino protein and formaldehyde groups with sodium cyanoborohydride (S Gidley et al., Biochem J. 1982, 203: 331 to 334; Jentoft et al. al.

Anal Biochem. 1980, 106: 186 to 190). By complexing residual inhibitory cyanide, the addition of nickel increases protein consumption during conjugation and leads to the formation of larger and potentially more immunogenic conjugates.

Differences in starting cyanide levels in sodium cyanoborohydride reagent lots also lead to inconsistent conjugation performance, resulting in variable product attributes such as conjugate size and ratio of Ps conjugated to CRM197. The addition of nickel reduced the conjugation inconsistency by complexing cyanide, eliminating differences in batches of sodium cyanoborohydride.

Suitable alternative chemicals include activating the saccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester.

The activated saccharide can thus be coupled directly or via a spacer (linker) group to an amino group on the carrier protein.

For example, the spacer can be cystamine or cysteamine to generate a thiolated polysaccharide that can be coupled to the carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g. using GMBS) or a carrier protein haloacetylated (e.g. using iodoacetimide [e.g. ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or SIAB, or SIA, or SBAP). Preferably, the cyanate ester (optionally produced by CDAP chemical) is coupled with hexane diamine or adipic acid dihydrazide (ADH) and the amino-derived saccharide is conjugated to the carrier protein using carbodiimide chemical (eg. e.g. EDAC or EDC) via a carboxyl group on the carrier protein.

Such conjugates are described in International Patent Application Publication WO 93/15760, WO 95/08348 and WO 96/29094; and Chu et al., 1983, Infect.

Immunity 40: 245 to 256. Other suitable conjugation techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.

Many are described in International Patent Application Publication WO 98/42721. Conjugation may involve a carbonyl linker which may be formed by reacting a free hydroxyl group on the saccharide with CDI (See Bethell et al., 1979, J. Biol. Chem. 254: 2572 a

2,574; Hearn et al., 1981, J. Chromatogr. 218: 509 to 518) followed by reaction with a protein to form a carbamate bond. This may involve reducing the anomeric terminus to a primary hydroxyl group, optionally protecting/deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a carbamate intermediate of CDI, and coupling the carbamate intermediate of CDI with an amino group. in a protein. After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-protein conjugates are purified (enriched for the amount of polysaccharide-protein conjugate) by one or more of a variety of techniques. Examples of such techniques are well known to those skilled in the art and include concentration/diafiltration, ultrafiltration, precipitation/elution, column chromatography and deep filtration operations. See, for example, US Patent 6,146,902. After the individual glycoconjugates are purified, they are compounded to formulate the immunogenic composition of the present invention. These pneumococcal conjugates can be prepared by separate processes and formulated by volume into a single dosage formulation. An alternative method for characterizing the glycoconjugates of the present invention is by the number of lysine residues in the carrier protein (e.g. CRM197) that become conjugated to the saccharide which can be characterized as a band of conjugated lysines (degree of conjugation). Evidence for lysine modification of the carrier protein, due to covalent bonds to polysaccharides, can be obtained by amino acid analysis using routine methods known to those skilled in the art. Conjugation results in a reduction in the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugated materials.

In a preferred embodiment, the extent of conjugation, as measured by lysine consumption of the glycoconjugate of the present invention, is between 2 and 15, between 2 and 13, between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 15, between 3 and 13, between 3 and 10, between 3 and 8, between 3 and 6, between 3 and 5, between 3 and 4, between 5 and 15, between 5 and 10, between 8 and 15, between 8 and 12, between 10 and 15, or between 10 and 12. In one embodiment, the degree of conjugation of the glycoconjugate of the present invention is about 2, about 3, about 4, about of 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In another embodiment, the degree of conjugation of the glycoconjugate of the present invention is between 4 and 7. In some embodiments, the carrier protein is CRM197. Glycoconjugates of the compositions of the present invention can also be characterized by the ratio (weight/weight) of polysaccharide to carrier protein (Ps:Pr). In some embodiments, the ratio of polysaccharide to glycoconjugate carrier protein (w/w) in the composition is between 0.5 and 3.0 (e.g., about 0.5, about 0.6, about 0.7 , about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2 .4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In other embodiments, the ratio of polysaccharide to carrier protein (w/w) is between 0.5 and 2.5, between 0.5 and 1.5, between 0.8 and 2.5, between 0.5 and 1 .0, between 1.0 and 1.5, between 1.0 and 2.0, between 0.8 and 2.4, between 0.8 and 2.3, between 0.8 and 2.2, between 0 .8 and 2.1, between 0.8 and 2.0, between 0.8 and 1.9, between 0.8 and 1.8, between 0.8 and 1.7, between 0.8 and 1, 6, between 0.8 and 1.5, between 0.8 and 1.4, between 0.8 and 1.3, between 0.9 and 2.4, between 0.9 and 2.3, between 0, 9 and 2.2, between 0.9 and

2.1, between 0.9 and 2.0, between 0.9 and 1.9, between 0.9 and 1.8, between 0.9 and 1.7, between 0.9 and 1.6, between 0.9 and 1.5, between 0.9 and 1.4, between 0.9 and 1.3, between 0.9 and 1.2, between 1.0 and 2.4, between 1.0 and 2 .3, between 1.0 and 2.2, between 1.0 and 2.1, between 1.0 and 2.0, between 1.0 and 1.9, between 1.0 and 1.8, between 1 .0 and 1.7, between 1.0 and 1.6, between 1.0 and 1.5, between 1.0 and 1.4, between 1.0 and 1.3 or between 1.0 and 1, two. In additional embodiments, the saccharide to carrier protein (w/w) ratio is between 0.8 and 1.2. In some such embodiments, the carrier protein is CRM197. The glycoconjugates and immunogenic compositions of the present invention may contain free saccharide that is not covalently conjugated to the carrier protein, but is nevertheless present in the glycoconjugate composition.

The free saccharide may be non-covalently associated (e.g., non-covalently linked to, adsorbed to, or attached to or with) the glycoconjugate.

In specific embodiments, the saccharide to carrier protein (w/w) ratio for the serotype 15A conjugate is from about 1.0 to about 2.0, from about 1.25 to about 1.75, or from about 1.3 to about 1.7. In other embodiments, the saccharide to carrier protein (w/w) ratio for serotype 15A is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1, 7 or 1.8. In specific embodiments, the saccharide to carrier protein (w/w) ratio for the conjugated serotype 15C is from about 1.0 to about 2.0, from about 1.25 to about 1.75, or from about 1.0 to about 1.75. about 1.3 to about 1.7. In other embodiments, the saccharide to carrier protein (w/w) ratio for serotype 15C is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1, 7 or 1.8. In specific embodiments, the saccharide to carrier protein (w/w) ratio for the conjugated 33F serotype is from about 1.0 to about 2.0, from about 1.25 to about 1.75, or from about 1.0 to about 1.75. about 1.3 to about 1.7. In other embodiments, the saccharide to carrier protein (w/w) ratio for serotype 33F is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1, 7 or 1.8.

In specific embodiments, the saccharide to carrier protein (w/w) ratio for the conjugated serotype 35B is from about 1.25 to about 2.25, from about 1.25 to about 2.0, or from about 1.25 to about 2.0. about 1.3 to about 1.8. In other embodiments, the saccharide to carrier protein (w/w) ratio for serotype 35B is about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1, 8, 1.9 or 2.0. In specific embodiments, the saccharide to carrier protein (w/w) ratio for the conjugated 24F serotype is from about 0.5 to about 1.5, from about 0.75 to about 1.25, or from about 0.75 to about 1.25. about 0.8 to about 1.0. In other embodiments, the saccharide to carrier protein (w/w) ratio for serotype 24F is about 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0. In a preferred embodiment, the glycoconjugate composition comprises less than about 50%, 45%, 40%, 35%, 30%, 25%, 20% or 15% free polysaccharide compared to the total amount of polysaccharide.

In a preferred embodiment, the glycoconjugate composition comprises less than about 25% free polysaccharide compared to the total amount of polysaccharide.

In a preferred embodiment, the glycoconjugate composition comprises less than about 20% free polysaccharide compared to the total amount of polysaccharide.

In a preferred embodiment, the glycoconjugate composition comprises less than about 15% free polysaccharide compared to the total amount of polysaccharide.

IV.

METHODS OF USE Embodiments of the present invention also include one or more of the multivalent immunogenic compositions described in the present invention (i) for use in, (ii) for use as a medicament or composition for, or (iii) for use in the preparation of a medicine for: (a) therapy (eg, of the human body); (b) medicine; (c) inhibition of infection with Streptococcus pneumoniae; (d) inducing an immune response or a protective immune response against S. pneumoniae; (e) prophylaxis of S. pneumoniae infection; (f) prevention of recurrence of S. pneumoniae infection; (g) reducing the progression, establishment or severity of pathological symptoms associated with S. pneumoniae infection including preventing associated complications such as brain damage, hearing loss and seizures, (h) reducing the likelihood of a S. pneumoniae infection or, (i) treatment, prophylaxis of or delay in the establishment, severity or progression of pneumococcal disease (or diseases), including, but not limited to: pneumococcal disease, pneumococcal bacteremia, pneumococcal meningitis, otitis media and sinusitis.

In such uses, the multivalent pneumococcal polysaccharide conjugate compositions of the present invention may optionally be employed in combination with one or more adjuvants, or without an adjuvant.

Accordingly, the present invention provides methods for the prophylactic treatment of (i.e., protection against) S. pneumoniae infection or pneumococcal disease comprising administering one or more of the multivalent immunogenic pneumococcal protein-polysaccharide conjugate compositions of the present invention to a patient. in need of treatment.

The compositions and formulations of the present invention can be used to protect or treat a human susceptible to infection, for example, a pneumococcal infection, by administering such a composition or formulation via a systemic or mucosal route.

In one embodiment, the present invention provides a method of inducing an immune response to S. pneumoniae, comprising administering to a patient an immunologically effective amount of a multivalent immunogenic composition of the present invention.

In another embodiment, the present invention provides a method of vaccinating a human against a pneumococcal infection, comprising the step of administering to the human an immunologically effective amount of a multivalent immunogenic composition of the present invention.

Thus, in one aspect, the present invention provides a method for (1) inducing an immune response in a human patient, (2) inducing a protective immune response in a human patient, (3) vaccinating a human patient against an infection with S. pneumoniae, or (4) reducing the likelihood of an S. pneumoniae infection in a human patient, the method comprising administering a multivalent immunogenic composition of the present invention to the patient (i.e., any multivalent immunogenic composition described in the present invention, such as the multivalent immunogenic compositions described in Section II entitled "Multivalent Immunogenic Compositions", supra). In one embodiment, the present invention provides a method for preventing pneumococcal pneumonia and/or invasive pneumococcal disease in a neonate (less than 1 year of age), infant (approximately 12 to 24 months), or young child (approximately 1 year of age). 2 to 5 years). In another embodiment, the present invention provides a method for preventing pneumococcal pneumonia and/or invasive pneumococcal disease in a patient from 6 weeks to 17 years of age.

In another embodiment, the present invention provides a method for preventing pneumococcal pneumonia and/or invasive pneumococcal disease in a patient aged 6 months to 17 years.

In another embodiment, the present invention provides a method for preventing pneumococcal pneumonia and/or invasive pneumococcal disease in adults 18 years of age and older.

In another embodiment, the present invention provides a method for preventing pneumococcal pneumonia and/or invasive pneumococcal disease in adults 50 years of age and older.

In another embodiment, the present invention provides a method for preventing pneumococcal pneumonia and/or invasive pneumococcal disease in adults 65 years of age and older.

In another embodiment, the present invention provides a method for preventing pneumococcal disease and/or invasive pneumococcal disease caused by one or more of the following strains of Streptococcus pneumoniae: 1, 3, 4, 5, 6A, 6B, 6C, 7F , 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

In one embodiment of the above methods, the composition comprises multiple S. pneumoniae protein-polysaccharide conjugates wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise serotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B or serotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F , 24F, 33F and 35B.

In another embodiment of the above methods, the composition comprises multiple polysaccharide-protein conjugates from S. pneumoniae wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes consist of serotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B or serotypes: 1, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

In one embodiment of the above methods, the composition comprises multiple S. pneumoniae protein-polysaccharide conjugates wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B or serotypes : 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

In another embodiment of the above methods, the composition comprises multiple polysaccharide-protein conjugates from S. pneumoniae wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes consist of serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B or serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

In one embodiment of the above methods, the composition comprises multiple S. pneumoniae protein-polysaccharide conjugates wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

In another embodiment of the above methods, the composition comprises multiple polysaccharide-protein conjugates from S. pneumoniae wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes consist of serotypes: 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B or serotypes: . A pneumococcal conjugate vaccine comprising serotype 6A polysaccharide has been shown to provide some cross-protection against serotype 6C ( Cooper et al., Vaccine 29 (2011) 7207 to 7211 ).

Therefore, in some embodiments of the above methods, the present invention also provides use of multivalent immunogenic compositions that do not comprise serotype 6C polysaccharide conjugate, but instead comprise serotype 6A polysaccharide conjugate or serotype 6A and 6B polysaccharide conjugates.

In other embodiments, the immunogenic composition comprises pneumococcal polysaccharide conjugates of serotypes 6A, 6B, and 6C.

In particular embodiments of the above methods, the multivalent immunogenic compositions comprise pneumococcal conjugates that include polysaccharides from a group of S. pneumoniae serotypes selected from the group consisting of: a) 1, 3, 4, 5, 6A, 6B, 7F , 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B;

i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; q) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; s) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,

23F, 24F, 33F and 35B; w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; and aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B.

In additional embodiments of the above methods, the composition comprises multiple S. pneumoniae protein-polysaccharide conjugates wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, wherein the S. pneumoniae serotypes comprise serotypes: i) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; or iii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B ; or iv) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B .

A pneumococcal conjugate vaccine comprising serotype 10A polysaccharide has also been shown to provide some cross-protection against serotype 39 (see WO 2017/085586). Therefore, in some embodiments of the above methods, the present invention also provides use of multivalent immunogenic compositions that do not comprise serotype 10A polysaccharide conjugate, but instead comprise serotype 39 polysaccharide conjugate. In other embodiments, the immunogenic composition comprises conjugates of pneumococcal polysaccharide serotypes 10A and 39. In particular embodiments of the above methods, the S. pneumoniae serotypes comprise a group of serotypes selected from the group consisting of: bb) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; cc) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; dd) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ff) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; gg) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; hh) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; kk) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ll) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39;

mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; nn) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; oo) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; pp) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; qq) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; rr) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; tt) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; uu) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; vv) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ww) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; zz) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B,

23F, 24F, 33F, 35B and 39; aaa) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; and bbb) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39. Shown It is also believed that immunogenic conjugates comprising S. pneumoniae serotype 15B capsular polysaccharide covalently linked to a carrier protein may provide some cross-protection against serotype 15C and/or serotype 15A (see WO 2015/110942). Therefore, in some embodiments of the above methods, the present invention also provides use of multivalent immunogenic compositions that do not comprise serotype 15C (or 15B de-O-acetylated) polysaccharide conjugate, but instead comprise serotype 15B polysaccharide conjugate ( i.e., the serotype 15B polysaccharide is not substantially de-O-acetylated). In other embodiments, the immunogenic composition comprises pneumococcal polysaccharide conjugates of serotypes 15B and 15C (or de-O-acetylated 15B). The compositions of the present invention are useful in methods of providing complementary protection against S. pneumoniae in patients who have previously received a multivalent pneumococcal vaccine.

In this use, the compositions of the present invention may provide protection against particular serotypes of S. pneumoniae that a patient has not previously been vaccinated against, may provide additional protection against serotypes of S. pneumoniae that a patient has previously been vaccinated against, or may provide protection against both S. pneumoniae serotypes that a patient has not previously been vaccinated against and S. pneumoniae serotypes that a patient has previously been vaccinated against.

Accordingly, the present invention provides a method of inducing an immune response, vaccinating, or inducing a protective immune response against S. pneumoniae in a patient, comprising administering a multivalent immunogenic composition to the patient, the composition comprising multiple polysaccharide-S protein conjugates. pneumoniae, wherein the polysaccharide-protein conjugates comprise capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein the patient has previously been vaccinated against S. pneumoniae.

In embodiments of this aspect of the present invention, the multivalent immunogenic composition can be any multivalent immunogenic composition described in the present invention.

In particular embodiments of the methods of the present invention, the multivalent immunogenic composition is administered to a patient who has previously been treated with a multivalent pneumococcal vaccine.

The multivalent immunogenic vaccine can be any vaccine that is indicated for the prevention of pneumococcal disease caused by more than one serotype of S. pneumoniae.

In specific embodiments of the above method, the patient has previously been treated with a multivalent pneumococcal vaccine that is indicated for the prevention of pneumococcal disease caused by one or more serotypes of S. pneumoniae in a group of serotypes selected from the group consisting of: i. 4, 6B, 9V, 14, 18C, 19F and 23F; ii. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F and 19A; iii. 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F; iv. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F and 33F; v. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20; and saw. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F, 8, 10A, 11A, 12F and 15B.

In specific embodiments of the above method, the multivalent pneumococcal vaccine comprises multiple polysaccharide-protein conjugates, wherein the polysaccharide-protein conjugates comprise polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein.

In other embodiments, the multivalent pneumococcal vaccine comprises multiple S. pneumoniae capsular polysaccharides that are not conjugated to a carrier protein.

In additional embodiments of the above method, the patient was previously treated with PREVNAR® 13 (Pneumococcal 13-valent Conjugate Vaccine [Diphtheric CRM197 Protein], Pfizer, Inc., Philadelphia, PA, USA). In additional embodiments of the above method, the patient was previously treated with PNEUMOVAX® 23 (Pneumococcal Polyvalent Vaccine, Merck & Co., Inc., Kenilworth, NJ, USA). In still further embodiments of the above method, the patient has previously been treated with SYNFLORIX™ (Pneumococcal Polysaccharide Conjugate Vaccine (adsorbed), GlaxoSmithKline Biologicals s.a., Rixensart, Belgium). In embodiments of the above method, the multivalent immunogenic composition of the present invention is administered to a patient at any time after the patient has received a multivalent pneumococcal vaccine, in accordance with the treatment regimen provided by the medical professional, e.g., a physician.

In particular embodiments, the multivalent immunogenic composition of the present invention is administered to a patient from about 1 month to about 5 years after the patient has received the multivalent pneumococcal vaccine, alternatively, from 1 month to 1 year, from 1 month to 2 years 1 month to 3 years 1 month to 4 years 1 month to 6 months 2 months to 6 months 2 months to 1 year 1 year to 5 years 6 months to 5 years , 6 months to 4 years, 6 months to 3 years, 6 months to 2 years, 6 months to 1 year, 1 year to 4 years, 1 year to 3 years, or 1 year to 2 years , after the patient has received the multivalent pneumococcal vaccine.

In additional embodiments, the multivalent immunogenic composition is administered to the patient about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months. months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.25 years, about 1.5 years, about 1.75 years, about 2 years, about 2.25 years, about 2.5 years, about 2.75 years, about 3 years, about 3.25 years, about 3.5 years, about 3.75 years, about 4 years, about 4.25 years, about 4.5 years, about 4.75 years, or about 5 years after the patient received the multivalent pneumococcal vaccine.

In additional embodiments, the present invention provides a method for (1) inducing an immune response in a human patient, (2) inducing a protective immune response in a human patient, (3) vaccinating a human patient against an infection with S. pneumoniae , or (4) reducing the likelihood of an S. pneumoniae infection in a human patient, the method comprising administering a multivalent immunogenic composition of the present invention and administering a multivalent pneumococcal vaccine to the patient, in either order.

For example, the patient is administered a multivalent pneumococcal vaccine first, and the patient is administered a multivalent immunogenic composition of the present invention thereafter.

Alternatively, the patient is administered a multivalent immunogenic composition of the present invention first and a multivalent pneumococcal vaccine is administered thereafter.

The multivalent pneumococcal vaccine can be any vaccine indicated for the prevention of pneumococcal disease caused by more than one S. pneumoniae serotype.

In specific embodiments of the above method, the patient is treated with a multivalent immunogenic composition of the present invention and a multivalent pneumococcal vaccine which is indicated for the prevention of pneumococcal disease caused by one or more serotypes of S. pneumoniae from a group of serotypes selected at from the group consisting of: i. 4, 6B, 9V, 14, 18C, 19F and 23F; ii. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F and 19A; iii. 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F; iv. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F and 33F; v. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20; and saw. 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 6A, 7F, 19A, 22F, 33F, 8, 10A, 11A, 12F and 15B.

In specific embodiments of the above method, the multivalent pneumococcal vaccine comprises capsular polysaccharides from S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 3, 5, 7F, 19A, 22F, 33F, 2, 8, 9N, 10A, 11A, 12F, 15B, 17F and 20A.

In specific embodiments of the above method, the multivalent pneumococcal vaccine comprises multiple polysaccharide-protein conjugates, wherein the polysaccharide-protein conjugates comprise polysaccharide of a serotype of S. pneumoniae conjugated to a carrier protein.

In other embodiments, the multivalent pneumococcal vaccine comprises multiple S. pneumoniae capsular polysaccharides that are not conjugated to a carrier protein.

In additional embodiments of the above method, the patient is treated with a multivalent immunogenic composition of the present invention and is treated with PREVNAR® 13 (13-valent Pneumococcal Conjugate Vaccine

[Diphtheria CRM197 Protein], Pfizer, Inc., Philadelphia, PA, USA), in any order.

In one embodiment, the patient is administered PREVNAR® 13 first, and the patient is administered a multivalent immunogenic composition of the present invention thereafter.

In alternative embodiments, the patient is administered a multivalent immunogenic composition of the present invention first and PREVNAR® 13 is administered thereafter.

In additional embodiments of the above method, the patient is treated with a multivalent immunogenic composition of the present invention and is treated with PNEUMOVAX® 23 (polyvalent pneumococcal vaccine, Merck & Co., Inc., Kenilworth, NJ, USA), in any order.

In one embodiment, the patient is administered PNEUMOVAX ® 23 first, and the patient is administered a multivalent immunogenic composition of the present invention thereafter.

In alternative embodiments, the patient is administered a multivalent immunogenic composition of the present invention first and PNEUMOVAX ® 23 is administered thereafter.

In still further embodiments of the above method, the patient is treated with a multivalent immunogenic composition of the present invention and is treated with SYNFLORIX™ (Pneumococcal Polysaccharide Conjugate Vaccine (adsorbed), GlaxoSmithKline Biologicals S.a., Rixensart, Belgium), in any order.

In one embodiment, the patient is administered SYNFLORIX™ first, and the patient is administered a multivalent immunogenic composition of the present invention thereafter.

In an alternative embodiment, the patient is administered a multivalent immunogenic composition of the present invention first and SYNFLORIX™ is administered thereafter.

In some embodiments of the above method, the multivalent immunogenic composition and the multivalent pneumococcal vaccine are administered simultaneously.

As used in the present invention, "simultaneous administration" is not limited to dosing two compositions at the same time, but includes administering one immediately after the other in any order.

In some embodiments, the multivalent immunogenic composition and the multivalent pneumococcal vaccine are administered via intramuscular or subcutaneous administration at separate anatomical sites, for example, two different arms.

In some embodiments of the above method, the amount of time between administration of the multivalent immunogenic composition of the present invention and the multivalent pneumococcal vaccine is from about 4 weeks to about 1 year.

In alternative embodiments, the amount of time is from about 1 month to about 5 years.

In one embodiment, the patient is administered the multivalent pneumococcal vaccine first and the multivalent immunogenic composition of the present invention thereafter.

In alternative embodiments, the patient is administered a multivalent immunogenic composition of the present invention first and the multivalent pneumococcal vaccine is administered thereafter.

Also provided is a method of inducing an immune response, vaccinating or inducing a protective immune response against S. pneumoniae in a patient, comprising: (1) administering a multivalent immunogenic composition of the present invention to the patient, (2) waiting for a predetermined amount of time to pass, and (3) administering a multivalent pneumococcal vaccine to the patient.

In such a method, the multivalent immunogenic composition may comprise any combination of S. pneumoniae protein-polysaccharide conjugates set forth in the present invention, and the multivalent pneumococcal vaccine may be any vaccine indicated for the prevention of disease caused by more than one S. pneumoniae serotype. .

Also provided by the present invention is a method of inducing an immune response, vaccinating or inducing a protective immune response against S. pneumoniae in a patient, comprising: (1) administering a multivalent pneumococcal vaccine to the patient, (2) waiting for a predetermined amount of time to elapse, and (3) administering a multivalent immunogenic composition of the present invention to the patient.

In such a method, the multivalent immunogenic composition may comprise any combination of S. pneumoniae protein-polysaccharide conjugates set forth in the present invention, and the multivalent pneumococcal vaccine may be any vaccine indicated for the prevention of disease caused by more than one S. pneumoniae serotype. .

In some embodiments of the above methods, the multivalent pneumococcal vaccine comprises multiple polysaccharide-protein conjugates from S. pneumoniae, wherein the polysaccharide-protein conjugates comprise capsular polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein.

In alternative embodiments, the multivalent pneumococcal vaccine comprises capsular polysaccharides from S. pneumoniae that are not conjugated to a carrier protein.

In any of the embodiments of the methods of the present invention (i.e., any of the methods described in the present invention), the method may further comprise administering one or more additional doses of a multivalent immunogenic composition of the present invention to the patient.

In such methods, the patient may have already received a multivalent pneumococcal vaccine prior to receiving a first dose of a multivalent immunogenic composition of the present invention, supra, or may not have been vaccinated against S. pneumoniae prior to receiving a multivalent immunogenic composition of the present invention. invention.

Thus, in one embodiment, a patient who has received a multivalent pneumococcal vaccine indicated for the prevention of pneumococcal disease caused by S. pneumoniae is administered two or more doses of a multivalent immunogenic composition of the present invention.

In alternative embodiments, a patient who has not previously been treated with any vaccine indicated for the prevention of pneumococcal disease is administered two or more doses of a multivalent immunogenic composition of the present invention.

In embodiments of the above method, the two or more doses are of the same multivalent immunogenic composition of the present invention.

In alternative embodiments, the two or more doses are of different multivalent immunogenic compositions of the present invention.

In specific embodiments of any of these methods, the patient is administered two, three or four doses of a multivalent immunogenic composition of the present invention.

In particular embodiments, the patient is immunocompromised (eg, is on an immunosuppressive regimen following a stem cell transplant). In some embodiments, the amount of time between administration of each dose of the multivalent immunogenic composition of the present invention is from about 4 weeks to about 1 year.

In the alternative embodiment, the amount of time between administration of each dose of the multivalent immunogenic composition of the present invention is from about 1 month to about 5 years.

In embodiments of any of the methods of the present invention, the patient to be treated with the composition (or compositions) of the present invention is a human.

In certain embodiments, the human patient is a newborn (approximately 6 weeks to 12 months). In certain embodiments, the human patient is an infant (approximately 12 to 24 months), or young child (approximately 2 to 5 years). The compositions of this present invention are also suitable for use with older children, adolescents and adults (e.g., 18 to 45 years of age, 18 to 50 years of age, 18 to 55 years of age, 18 to 60 years or 18 to 65 years). In other embodiments of any of the methods of the present invention, the patient is from about 2 to about 18 years of age.

In additional embodiments of any of the methods of the present invention, the patient is 18 years of age or older.

In additional embodiments of the methods of the present invention, the patient is a neonate and the neonate is administered 1, 2 or 3 doses of a multivalent immunogenic composition of the present invention.

The amount of time between administration of each dose may vary, but an example of a dosing schedule includes administration of one dose at 2 months of age, then another administration of one dose at 4 months of age, and finally one administration end of a dose at 6 months of age.

Another example of an administration schedule in neonates is administration of one dose at 2 months of age and then another administration of one dose at 3 months of age.

Another example of an administration schedule in neonates is administration of one dose at 2 months of age, then another administration of one dose at 3 months of age, and finally a final administration of one dose at 6 months of age. .

In additional embodiments, a newborn patient may receive an additional "booster" dose of a multivalent immunogenic composition of the present invention when the newborn becomes an infant.

For example, a new

newborn receives the dose at 2 months of age, then another administration of one dose at 4 months of age, and finally a final administration of one dose at 6 months of age, then when the newborn reaches the age of a baby , an additional "booster" dose of a multivalent immunogenic composition of the present invention is administered between 11 to 15 months of age.

In one embodiment, a newborn is administered 2 doses of a multivalent immunogenic composition of the present invention.

In one embodiment, a newborn is administered 3 doses of a multivalent immunogenic composition of the present invention.

In one embodiment, a patient is administered 3 doses of a multivalent immunogenic composition of the present invention, wherein the first and second doses are administered between 2 and 10 months of age and the third dose is administered between 11 to 15 months of age.

In one embodiment, a patient is administered 4 doses of a multivalent immunogenic composition of the present invention, wherein the first dose is administered at 2 months of age, the second dose is administered at 4 months of age, the third dose is administered at 2 months of age. 6 months of age and the fourth dose is given between 11 to 15 months of age.

In additional embodiments of the methods of the present invention, the human patient is elderly.

In some embodiments of any of the methods of the present invention, the patient is 50 years of age or older.

In some embodiments of any of the methods of the present invention, the patient is 55 years of age or older.

In some embodiments of any of the methods of the present invention, the patient is 60 years of age or older.

In still further embodiments of any of the methods of the present invention, the patient is 65 years of age or older.

In additional embodiments of any of the methods of the present invention, the patient is 70 years of age or older.

In some embodiments of any of the methods of the present invention, the patient to be treated with an immunogenic composition of the present invention is immunocompromised.

In some embodiments of any of the methods of the present invention, the multivalent immunogenic composition of the present invention is administered concurrently with an influenza vaccine.

In certain embodiments, the influenza vaccine is an “elderly flu shot,” a high-dose flu vaccine intended for the elderly, for example, persons aged 65 and older.

The present invention provides a method of inducing a protective immune response in a patient against a pneumococcal infection comprising the step of administering to the patient an immunologically effective amount of any of the multivalent immunogenic pneumococcal protein-polysaccharide conjugate compositions described in the present invention.

Optimal amounts of components for a particular vaccine (e.g., a multivalent immunogenic composition of the present invention) can be determined by standard studies that involve observation of adequate immune responses in subjects.

For example, in another embodiment, the dosage for human vaccination is determined by extrapolation from animal studies to human data.

In another embodiment, the dosage is determined empirically.

The methods of the present invention can be used for the prevention and/or reduction of primary clinical syndromes caused by microbes, for example, S. pneumoniae, including both invasive infections (meningitis, pneumonia and bacteremia) and non-invasive infections (acute otitis media and sinusitis).

Administration of the compositions of the present invention may include one or more of: injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/food, respiratory or genitourinary tracts.

In one embodiment, intranasal administration is used for the treatment of pneumonia or otitis media (as nasopharyngeal transport of pneumococcal infections can be more effectively prevented, thereby attenuating infection at its earliest stage). In specific embodiments, the compositions of the present invention are administered to the patient via intramuscular or subcutaneous administration.

All publications mentioned in the present invention are incorporated by reference for purposes of describing and disclosing methodologies and materials that may be used in connection with the present invention.

As different embodiments of the present invention are described in the present invention with reference to the accompanying drawings, it should be understood that the present invention is not limited to those precise embodiments, and that various changes and modifications may be made thereto by one skilled in the art without departing from the scope or spirit of the present invention, as defined in the appended claims.

The following examples illustrate, but do not limit, the present invention.

EXAMPLE 1 Preparation of Capsular Polysaccharides from S. pneumoniae Methods for culturing pneumococci are well known in the art.

See, for example, Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods for preparing pneumococcal capsular polysaccharides are also well known in the art.

See, for example, European Patent EP 0 497 524 B1. The process described below generally follows the method described in European Patent EP 0 497 524 B1 and is generally applicable to all pneumococcal serotypes.

Pneumococcal strain isolates for serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23F, 33F and 35B were obtained from the Merck Culture Collection.

Strains for serotypes 23B were obtained from the Centers for Disease Control and Prevention and the University of Alabama, Birmingham.

Strains for serotype 24F were obtained from Merck Culture Collection and University of Alabama, Birmingham.

When necessary, subtypes were differentiated based on the Quellung reaction using specific antisera.

See, for example, US patent 5,847,112. The isolates obtained were additionally clonally isolated by serial plating in two stages on agar plates consisting of an animal component-free medium containing soy peptone, yeast extract and glucose without hemin.

For serotype 7F, the agar plates used also contained hemin.

Clonal isolates for each serotype were further expanded in liquid culture using animal component-free media containing soy peptone, yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate, glucose, and glycerol to prepare the cell banks. pre-majors.

Production of each pneumococcal polysaccharide serotype consisted of a cell expansion and batch production fermentation followed by chemical inactivation before downstream purification.

A thawed cell bank ampoule from each serotype was expanded using a shaken culture flask or bottle containing a pre-sterilized animal component-free growth medium containing soy peptone or soy peptone ultrafiltrate, yeast extract, or yeast extract, HEPES, sodium chloride, sodium bicarbonate, potassium phosphate and glucose.

The cell expansion culture was grown in a sealed shaker flask or bottle to minimize gas exchange with controlled temperature and agitation.

During the cell expansion of these serotypes, temperature, pH, pressure and agitation were controlled.

Airflow overlap was also controlled as spray was not used.

After obtaining a specified culture density, as measured by optical density at 600 nm, a portion of the cell expansion culture was transferred to a production fermenter containing pre-sterilized animal-free growth medium containing soy peptone or ultrafiltered. soy peptone, yeast extract or yeast extract ultrafiltrate, sodium chloride, potassium phosphate and glucose.

Temperature, pH, pressure and agitation were controlled.

Airflow overlap was also controlled as spray was not used.

Batch fermentation was terminated by the addition of a chemical inactivating agent, phenol, when glucose was nearly exhausted.

Pure phenol was added to a final concentration of 0.8 to 1.2% to inactivate the cells and release the capsular polysaccharide from the cell wall.

Primary inactivation takes place for a specific time in the fermenter where temperature and agitation continue to be controlled.

After primary inactivation, the batch was transferred to another container where it was kept for a specific additional time at controlled temperature and agitation for complete inactivation.

This was confirmed by microbial plating techniques or by checking the phenol concentration and specific time.

The inactivated broth was then purified.

EXAMPLE 2

Purification of Pneumococcal Polysaccharides The purification process for pneumococcal polysaccharides consisted of several steps of centrifugation, deep filtration, concentration/diafiltration and precipitation operations.

All procedures were performed at room temperature, unless otherwise specified.

Inactivated broth from S. pneumoniae fermenter cultures was flocculated with a cationic polymer (such as BPA-1000, TRETOLITE® (Baker Hughes Inc., Houston, TX), Spectrum 8160, poly(ethyleneimine), and Millipore pDADMAC). Cationic polymers bound to impurity proteins, nucleic acids and cellular residues.

After the flocculation step and an aging period, the flocculated solids were removed through centrifugation steps and multiple deep filtrations.

The clarified broth was concentrated and diafiltered using a 100 kDa to 500 kDa MWCO filter (molecular weight cut-off). Diafiltration was achieved using Tris, MgCl2 buffer and sodium phosphate buffer.

Diafiltration removed residual nucleic acid and protein.

Removal of additional impurities was achieved by reprecipitating the polysaccharide in sodium acetate and phenol with alcohol and/or denatured isopropanol.

During the phenol precipitation step, sodium acetate in sodium phosphate saline buffer and phenol (liquefied phenols or solid phenols) were loaded into the diafiltered retentate.

The alcohol fractionation of the polysaccharide was then carried out in two stages.

In the first stage a low percentage alcohol was added to the preparation to precipitate cellular debris and other unwanted impurities while the crude polysaccharide remained in solution.

Impurities were removed by centrifugation followed by a deep filtration step.

The polysaccharide was then recovered from the solution by adding additional isopropanol or denatured alcohol to the batch.

The precipitated polysaccharide pellet was recovered by centrifugation, ground and dried as a powder and stored frozen at -70°C.

EXAMPLE 3 Preparation of Serotype 1 Conjugate for Polyvalent Study of PCV23 (DMSO) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in dimethylsulfoxide (DMSO). The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by dialysis before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 25,000 kPa/5 passes (250 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 15 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 2.5 mg Ps/mL with a sucrose concentration of 10% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 1.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was dialyzed at approximately 4°C for 3 days against 150 mM sodium chloride, 0.05% (w/v) polysorbate 20 using a 300 kDa NMWCO dialysis cassette.

Final Filtration and Product Storage The batch was 0.2 micron filtered (with 0.5 micron pre-filter), aliquoted and frozen at ≤ −60°C.

EXAMPLE 4 Preparation of Serotype 1 Conjugate for Polyvalent Study of PCV23 (DMSO + Aq) Using Aqueous Conjugation The polysaccharide was dissolved, size reduced, chemically activated and buffer exchanged by ultrafiltration.

The purified CRM197 was then conjugated to the activated polysaccharide using the nickel chloride in the reaction mixture, and the resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water, and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular weight.

The homogenization pressure and number of passes through the homogenizer were controlled at 25,000 kPa/5 passes (250 bar/5 passes) to reduce the size to a target molecular weight.

The size-reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 15 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5M potassium phosphate, pH 7.0. The pH of the buffer selected was to improve the stability of activated polysaccharide during the conjugation reaction.

Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was combined with the buffered polysaccharide solution at a mass ratio of polysaccharide to CRM197 of 0.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Polysaccharide and phosphate concentrations were 6.9 g/L and 100 mM, respectively.

The polysaccharide concentration was selected to control the size of the resulting conjugate.

Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution.

Sodium cyanoborohydride (2 mol per mol of polysaccharide repeat unit) was added.

Conjugation continued for 120 hours to maximize polysaccharide and protein consumption.

Reduction with Sodium Borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g/L, cooled to 2 to 8 °C, and filtered at 1.2 micron.

The batch was diafiltered against 100 mM potassium phosphate, pH 7.0 at 2 at 8°C using a 100 kDa NMWCO tangential flow ultrafiltration membrane.

The batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide/L and pH adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mol of polysaccharide repeat unit) was added. 1.5M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

Polysorbate 20 was added to the retentate batch at a concentration of 0.05% (w/v), then the batch was filtered at 0.2 micron.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer with 0.015% (w/v) polysorbate 20. The batch was dispensed in aliquots and frozen at ≤ −60 °C.

EXAMPLE 5 Preparation of Serotype 1 Conjugate for Polyvalent Study of PCV22 Using Aqueous Conjugation Polysaccharide was dissolved, size reduced, chemically activated and buffer exchanged by ultrafiltration.

The purified CRM197 was then conjugated to the activated polysaccharide using the nickel chloride in the reaction mixture, and the resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water, and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular weight.

The homogenization pressure and number of passes through the homogenizer were controlled at 25,000 kPa/5 passes (250 bar/5 passes) to reduce the size to a target molecular weight.

The size-reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 15 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5M potassium phosphate, pH 7.0. The pH of the buffer selected was to improve the stability of activated polysaccharide during the conjugation reaction.

Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was combined with the buffered polysaccharide solution at a mass ratio of polysaccharide to CRM197 of 0.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Polysaccharide and phosphate concentrations were 6.9 g/L and 100 mM, respectively.

The polysaccharide concentration was selected to control the size of the resulting conjugate.

The solution was then filtered through 0.2 micron.

Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution.

Sodium cyanoborohydride (2 mol per mol of polysaccharide repeat unit) was added.

Conjugation continued for 120 hours to maximize polysaccharide and protein consumption.

Reduction with Sodium Borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g/L, cooled to 2 to 8 °C, and filtered at 1.2 micron.

The batch was diafiltered against 100 mM potassium phosphate, pH 7.0 at 2 at 8°C using a 100 kDa NMWCO tangential flow ultrafiltration membrane.

The batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide/L and pH adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mol of polysaccharide repeat unit) was added. 1.5M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

Polysorbate 20 was added to the retentate batch at a concentration of 0.05% (w/v), then the batch was filtered at 0.2 micron.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer with 0.015% (w/v) polysorbate 20. The batch was dispensed in aliquots and frozen at ≤ −60 °C.

EXAMPLE 6 Preparation of Serotype 3 Conjugate for Polyvalent Study of PCV23 (DMSO) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in dimethylsulfoxide (DMSO). The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 81,000 kPa/6 passes (810 bar/6 passes) followed by 90,000 kPa/3 passes (900 bar/3 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 12 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 2.5 mg Ps/mL with a sucrose concentration of 10% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 2.25 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.35. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, aliquoted and frozen at ≤ −60 °C.

EXAMPLE 7 Preparation of Serotype 3 Conjugate for Polyvalent Studies of PCV22 and PCV23 (DMSO + Aq) Using Aqueous Conjugation The polysaccharide was dissolved, size reduced, chemically activated and buffer exchanged by ultrafiltration.

The purified CRM197 was then conjugated to the activated polysaccharide using the nickel chloride in the reaction mixture, and the resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water, and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular weight.

The homogenization pressure and number of passes through the homogenizer were controlled at 38,000 kPa/5 passes (380 bar/5 passes) to reduce the size to a target molecular weight.

The size-reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 12 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5M potassium phosphate, pH 6.0. The pH of the buffer selected was to improve the stability of activated polysaccharide during the conjugation reaction.

Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was filtered at 0.2 micron and combined with the buffered polysaccharide solution at a mass ratio of polysaccharide to CRM197 of 0.6 . The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Polysaccharide and phosphate concentrations were 4.1 g/L and 150 mM, respectively.

The polysaccharide concentration was selected to control the size of the resulting conjugate.

The solution was then filtered through 0.2 micron.

Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution.

Sodium cyanoborohydride (2 mol per mol of polysaccharide repeat unit) was added.

Conjugation was continued for 120 hours at 10 °C to maximize polysaccharide and protein consumption.

Reduction with Sodium Borohydride

After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g/L, cooled to 2 to 8 °C, and filtered at 1.2 micron.

The batch was diafiltered against 100 mM potassium phosphate, pH 7.0 at 2 at 8°C using a 100 kDa NMWCO tangential flow ultrafiltration membrane.

The batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide/L and pH adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mol of polysaccharide repeat unit) was added. 1.5M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The batch was filtered at 0.2 micron.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer. The batch was aliquoted and frozen at ≤ −60 °C.

EXAMPLE 8 Preparation of Serotype 4 Conjugate for Polyvalent Study of PCV23 (DMSO) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by dialysis before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature,

concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 30,000 kPa/5 passes (300 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 50°C and pH 4.1 with a sodium acetate buffer to partially deketalize the polysaccharide.

The polysaccharide solution was then cooled to 22 °C before activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 4 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 5.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 2.0. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was dialyzed at approximately 4°C for 3 days against 150 mM sodium chloride, 0.05% (w/v) polysorbate 20 using a 300 kDa NMWCO dialysis cassette.

Final Filtration and Product Storage The batch was 0.2 micron filtered (with 0.5 micron pre-filter), aliquoted and frozen at ≤ −60°C.

EXAMPLE 9 Serotype 4 Conjugate Preparation for Polyvalent Studies of PCV23 (DMSO + Aq) and PCV22 Using Aqueous Conjugation

The polysaccharide was dissolved, reduced in size, chemically activated and buffer exchanged by ultrafiltration.

The purified CRM197 was then conjugated to the activated polysaccharide using the nickel chloride in the reaction mixture, and the resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water, and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular weight.

The homogenization pressure and number of passes through the homogenizer were controlled at 30,000 kPa/5 passes (300 bar/5 passes) to reduce the size to a target molecular weight.

The size-reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 50°C and pH 4.1 with a sodium acetate buffer to partially deketalize the polysaccharide.

The polysaccharide solution was then cooled to 22 °C before activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 4 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5M potassium phosphate, pH 7.0. The pH of the buffer selected was to improve the stability of activated polysaccharide during the conjugation reaction.

Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was filtered at 0.2 micron and combined with the buffered polysaccharide solution at a mass ratio of polysaccharide to CRM197 of 0.5 . The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Polysaccharide and phosphate concentrations were 8.3 g/L and 100 mM, respectively.

The polysaccharide concentration was selected to control the size of the resulting conjugate.

The solution was then filtered through 0.2 micron.

Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution.

Sodium cyanoborohydride (2 mol per mol of polysaccharide repeat unit) was added.

Conjugation continued for 120 hours to maximize polysaccharide and protein consumption.

Reduction with Sodium Borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g/L, cooled to 2 to 8 °C, and filtered at 1.2 micron.

The batch was diafiltered against 100 mM potassium phosphate, pH 7.0 at 2 at 8°C using a 100 kDa NMWCO tangential flow ultrafiltration membrane.

The batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide/L and pH adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mol of polysaccharide repeat unit) was added. 1.5M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

Then, the batch was filtered at 0.2 micron.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer. The batch was aliquoted and frozen at ≤ −60 °C.

EXAMPLE 10 Preparation of Serotype 5 Conjugate for Polyvalent Study of PCV23 (DMSO) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by dialysis before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 60,000 kPa/5 passes (600 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 4°C and pH 4.1 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 4 hours at 4°C.

The activated product was diafiltered against 10 mM Sodium Acetate, pH 4.1 using a 10 kDa NMWCO tangential flow ultrafiltration membrane, followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide solution was spiked with sodium chloride to a concentration of 20 mM.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 2.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added,

and conjugation proceeded at 22°C.

Dilution and Neutralization The batch was diluted in 150 mM sodium chloride with approximately 0.025 % (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was dialyzed at approximately 4°C for 3 days against 150 mM sodium chloride, 0.05% (w/v) polysorbate 20 using a 300 kDa NMWCO dialysis cassette.

Final Filtration and Product Storage The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), aliquoted and frozen at ≤ −60°C.

EXAMPLE 11 Preparation of Serotype 5 Conjugate for Polyvalent Studies of PCV22 and PCV23 (DMSO + Aq) Using Aqueous Conjugation The polysaccharide was dissolved, size reduced, chemically activated and buffer exchanged by ultrafiltration.

The purified CRM197 was then conjugated to the activated polysaccharide using the nickel chloride in the reaction mixture, and the resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water, and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular weight.

The homogenization pressure and number of passes through the homogenizer were controlled at 60,000 kPa/5 passes (600 bar/5 passes) to reduce the size to a target molecular weight.

The size-reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 4°C and pH 4.1 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 4 hours at 4°C.

The activated product was diafiltered against 10 mM sodium acetate, pH 4.1 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5M potassium phosphate, pH 6.0. The pH of the buffer selected was to improve the stability of activated polysaccharide during the conjugation reaction.

Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was combined with the buffered polysaccharide solution at a mass ratio of polysaccharide to CRM197 of 0.4. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Polysaccharide and phosphate concentrations were 3.8 g/L and 150 mM, respectively.

The polysaccharide concentration was selected to control the size of the resulting conjugate.

The solution was then filtered through 0.2 micron.

Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution.

Sodium cyanoborohydride (2 mol per mol of polysaccharide repeat unit) was added.

Conjugation continued for 96 hours to maximize polysaccharide and protein consumption.

Purification and neutralization

After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g/L, cooled to 2 to 8 °C, and filtered at 1.2 micron.

The batch was diafiltered against 300 mM sodium bicarbonate pH 9.3 at 2 at 8°C using a 100 kDa NMWCO tangential flow ultrafiltration membrane.

The batch, recovered in the retentate, was then neutralized with 1.5M potassium phosphate, pH 6.0. Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

Polysorbate 20 was added to the retentate batch at a concentration of 0.05% (w/v) and then the batch was filtered at 0.2 micron.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer with 0.015% (w/v) polysorbate 20. The batch was dispensed in aliquots and frozen at ≤ −60 °C.

EXAMPLE 12 Preparation of Serotype 6A Conjugate for Polyvalent Studies of PCV23 (DMSO) and PCV23 (DMSO+Aq) and PCV 22 Using DMSO Conjugation Polysaccharide was dissolved, chemically activated and buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 20,000 kPa/5 passes (200 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO of 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 1.5 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.4. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7 using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.025 % (w/v) polysorbate 20, dispensed in aliquots and frozen at ≤ −60°C.

EXAMPLE 13 Preparation of Serotype 6B Conjugate for Polyvalent Studies of PCV 23 (DMSO) and PCV 23 (DMSO/Aq) Using DMSO Conjugation

The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 20,000 kPa/5 passes (200 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO of 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 1.85 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.35. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a tangential flow ultrafiltration membrane of NMWCO of 300 kDa.

The retentate batch was 0.2 micron filtered, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.025 % (w/v) polysorbate 20, dispensed in aliquots and frozen at ≤ −60°C.

EXAMPLE 14 Preparation of Serotype 6B Conjugate for Polyvalent Study of PCV22 Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 20,000 kPa/5 passes (200 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 1.75 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.35. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride

Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.025 % (w/v) polysorbate 20, dispensed in aliquots and frozen at ≤ −60°C.

EXAMPLE 15 Preparation of Serotype 7F Conjugate for Polyvalent Studies of PCV23 (DMSO) and PCV23 (DMSO + Aq) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 15,000 kPa/7 passes (150 bar/7 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 4°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 4 hours at 4°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 2.6 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.025 % (w/v) polysorbate 20, dispensed in aliquots and frozen at ≤ −60°C.

EXAMPLE 16 Preparation of Serotype 7F Conjugate for Polyvalent Study of PCV22

Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer changed by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 15,000 kPa/7 passes (150 bar/7 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 4°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 4 hours at 4°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 2.04 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage

The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a tangential flow ultrafiltration membrane of NMWCO of 300 kDa.

The retentate batch was 0.2 micron filtered, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.025 % (w/v) polysorbate 20, dispensed in aliquots and frozen at ≤ −60°C.

EXAMPLE 17 Preparation of Serotype 8 Conjugate for Polyvalent Studies of PCV23 (DMSO) and PCV23 (DMSO + Aq) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 60,000 kPa/6 passes (600 bar/6 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 4 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 4.5 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

After mixing, the conjugation reaction proceeded at 22 °C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, aliquoted and frozen at ≤ −60 °C.

EXAMPLE 18 Preparation of Serotype 9V Conjugate for Polyvalent PCV23 Study (DMSO) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 23,000 kPa/5.5 passes (230 bar/5.5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 6 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of

3.0g Ps/L and a polysaccharide to CRM197 mass ratio of 1.3. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with a 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.025 % (w/v) ) of polysorbate 20, dispensed in aliquots and frozen at ≤ −60 °C.

EXAMPLE 19 Preparation of Serotype 9V Conjugate for Polyvalent Study of PCV22 Using Aqueous Conjugation Polysaccharide was dissolved, size reduced, chemically activated and buffer exchanged by ultrafiltration.

The purified CRM197 was then conjugated to the activated polysaccharide using the nickel chloride in the reaction mixture, and the resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water, and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular weight.

The homogenization pressure and number of passes through the homogenizer were controlled at 10,000 kPa/5 passes (100 bar/5 passes) to reduce the size to a target molecular weight.

The size-reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 6 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5M potassium phosphate, pH 7.0. The pH of the buffer selected was to improve the stability of activated polysaccharide during the conjugation reaction.

THE

Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was filtered at 0.2 micron and combined with the buffered polysaccharide solution at a mass ratio of polysaccharide to CRM197 of 0.7. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Polysaccharide and phosphate concentrations were 10.0 g/L and 100 mM, respectively.

The polysaccharide concentration was selected to control the size of the resulting conjugate.

The solution was then filtered through 0.2 micron.

Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution.

Sodium cyanoborohydride (2 mol per mol of polysaccharide repeat unit) was added.

Conjugation continued for 120 hours to maximize polysaccharide and protein consumption.

Reduction with Sodium Borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g/L, cooled to 2 to 8 °C, and filtered at 1.2 micron.

The batch was diafiltered against 100 mM potassium phosphate, pH 7.0 at 2 at 8°C using a 100 kDa NMWCO tangential flow ultrafiltration membrane.

The batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide/L and pH adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mol of polysaccharide repeat unit) was added. 1.5M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

Polysorbate 20 was added to the retentate batch at a concentration of 0.05% (w/v), then the batch was filtered at 0.2 micron.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer with 0.015% (w/v) polysorbate 20. The batch was dispensed in aliquots and frozen at ≤ −60 °C.

EXAMPLE 20 Preparation of Serotype 9V Conjugate for Polyvalent Study of PCV23 (DMSO + Aq) Using Aqueous Conjugation Polysaccharide was dissolved, size reduced, chemically activated and buffer exchanged by ultrafiltration.

The purified CRM197 was then conjugated to the activated polysaccharide using the nickel chloride in the reaction mixture, and the resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water, and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular weight.

The homogenization pressure and number of passes through the homogenizer were controlled at 10,000 kPa/5 passes (100 bar/5 passes) to reduce the size to a target molecular weight.

The size-reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 6 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5M potassium phosphate, pH 7.0. The pH of the buffer selected was to improve the stability of activated polysaccharide during the conjugation reaction.

Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was filtered at 0.2 micron and combined with the buffered polysaccharide solution at a mass ratio of polysaccharide to CRM197 of 0.7 . The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Polysaccharide and phosphate concentrations were 10.0 g/L and 100 mM, respectively.

The polysaccharide concentration was selected to control the size of the resulting conjugate.

The solution was then filtered through 0.2 micron.

Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution.

Sodium cyanoborohydride (2 mol per mol of polysaccharide repeat unit) was added.

Conjugation continued for 120 hours to maximize polysaccharide and protein consumption.

Reduction with Sodium Borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g/L, cooled to 2 to 8 °C, and filtered at 1.2 micron.

The batch was diafiltered against 100 mM potassium phosphate,

pH 7.0 at 2 at 8°C using a 100 kDa NMWCO tangential flow ultrafiltration membrane.

The batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide/L and pH adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mol of polysaccharide repeat unit) was added. 1.5M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

Polysorbate 20 was added to the retentate batch at a concentration of 0.05% (w/v), then the batch was filtered at 0.2 micron.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer with 0.015% (w/v) polysorbate 20. The batch was dispensed in aliquots and frozen at ≤ −60 °C.

EXAMPLE 21 Preparation of Serotype 10A Conjugate for Polyvalent Study of PCV23 Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 61,500 kPa/5 passes (615 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 3.5 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.6. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

Final filtration and product storage The batch was concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.025 % (w/v) polysorbate 20 at 4 °C using an ultrafiltration membrane of tangential flow of 300 kDa NMWCO.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, aliquoted and frozen at ≤ −60 °C.

EXAMPLE 22 Preparation of Serotype 10A Conjugate for Polyvalent Study of PCV23 (DMSO + Aq) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 60,000 kPa/5 passes (600 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 3.4 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.6. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

Final filtration and product storage The batch was then concentrated and diafiltered against 150 mM sodium chloride, 25 mM potassium phosphate, pH 7 followed by diafiltration against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.025% (w/v) polysorbate 20 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, aliquoted and frozen at ≤ −60 °C.

EXAMPLE 23 Preparation of Serotype 10A Conjugate for Polyvalent Study of PCV22 Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 60,000 kPa/5 passes (600 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 3.8 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.75. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C. Potassium phosphate buffer was then added to neutralize the pH. The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7 using a 30 kDa NMWCO tangential flow ultrafiltration membrane. Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration. The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, aliquoted and frozen at ≤ −60 °C.

EXAMPLE X Preparation of Serotype 11A Conjugate for Polyvalent Study of PCV24 Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration. Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO. The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration. Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes. Polysaccharide size reduction and oxidation

Purified pneumococcal Ps capsular powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 80,000 kPa/8 passes (800 bar/8 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide solutions and

CRM197 were blended to obtain a polysaccharide concentration of 2.3 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 24 Preparation of Serotype 12F Conjugate for Polyvalent Studies of PCV23 (DMSO) and PCV23 (DMSO + Aq) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was reduced in size by acid hydrolysis by adding 200 mM acetic acid, incubating at 80 °C for 155 minutes, then neutralizing by adding cold potassium phosphate buffer pH 7 to 400 mM.

The size-reduced polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 2.7 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.8. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 25 Preparation of Serotype 12F Conjugate for Polyvalent Studies of PCV23 (DMSO) and PCV23 (DMSO + Aq) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was reduced in size by acid hydrolysis by adding 200 mM acetic acid, incubating at 90°C for 60 minutes, then neutralizing by adding cold potassium phosphate buffer pH 7 to 400 mM.

The size-reduced polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 3.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7.0 using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 26 Preparation of Serotype 14 Conjugate for Polyvalent Study of PCV23 (DMSO) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by dialysis before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 20,000 kPa/6 passes (200 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 4 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide solutions and

CRM197 were blended to obtain a polysaccharide concentration of 1.8 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was dialyzed at approximately 4°C for 22.5 hours against 150 mM sodium chloride, 0.05% polysorbate 20 using a 300 kDa MWCO dialysis cassette.

Final Filtration and Product Storage The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), aliquoted and frozen at ≤ −60°C.

EXAMPLE 27 Preparation of Serotype 14 Conjugate for Polyvalent Study of PCV22 and PCV23 (DMSO + Aq) Using Aqueous Conjugation Polysaccharide was dissolved, size reduced, chemically activated and buffer exchanged by ultrafiltration.

The purified CRM197 was then conjugated to the activated polysaccharide using the nickel chloride in the reaction mixture, and the resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water, and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular weight.

The homogenization pressure and number of passes through the homogenizer were controlled at 20,000 kPa/6 passes (200 bar/6 passes) to reduce the size to a target molecular weight.

The size-reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 4 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5M potassium phosphate, pH 7.0. The pH of the buffer selected was to improve the stability of activated polysaccharide during the conjugation reaction.

Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was filtered at 0.2 micron and combined with the buffered polysaccharide solution at a mass ratio of polysaccharide to CRM197 of 1.0 . The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Polysaccharide and phosphate concentrations were 3.8 g/L and 100 mM, respectively.

The polysaccharide concentration was selected to control the size of the resulting conjugate.

The solution was then filtered through 0.2 micron.

Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution.

Sodium cyanoborohydride (2 mol per mol of polysaccharide repeat unit) was added.

Conjugation continued for 72 hours to maximize polysaccharide and protein consumption.

Reduction with Sodium Borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g/L, cooled to 2 to 8 °C, and filtered at 1.2 micron.

The batch was diafiltered against 100 mM potassium phosphate, pH 7.0 at 2 at 8°C using a 100 kDa NMWCO tangential flow ultrafiltration membrane.

The batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide/L and pH adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mol of polysaccharide repeat unit) was added. 1.5M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The batch was then filtered at 0.2 micron.

The batch was adjusted to a polysaccharide concentration of 1.0 g/L with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer. The batch was aliquoted and frozen at ≤ −60 °C.

EXAMPLE 28 Preparation of Serotype 15A Conjugate for Multipurpose Studies of

PCV23 (DMSO) and PCV23 (DMSO + Aq) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 21,000 kPa/5 passes (210 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 20 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO which were preheated to 34°C.

The polysaccharide solution was spiked with sodium chloride to a concentration of 25 mM.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 5.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 2.0. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 34°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 34°C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 29 Preparation of Serotype 15A Conjugate for Polyvalent Study of PCV22 Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 20,000 kPa/5 passes (200 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a tangential flow ultrafiltration membrane of

10 kDa NMWCO.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 20 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

Activated polysaccharides were formulated for lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO which were preheated to 34°C.

The polysaccharide solution was spiked with sodium chloride to a concentration of 25 mM.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 5.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 2.0. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 34°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 30 Preparation of Serotype 15C Conjugate for Polyvalent Studies of PCV23 (DMSO) and PCV23 (DMSO + Aq) Using DMSO Conjugation Streptococcus pneumoniae serotype 15B polysaccharide derivative was dissolved, sized to a target molecular weight, subjected to hydrolysis mild base to release O-acetyl groups, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction, Base Hydrolysis, and Oxidation Purified Ps capsular pneumococcal serotype 15B powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 30,000 kPa/5 passes (300 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was heated to 60°C and sodium bicarbonate pH9 buffer was added to a final concentration of 50 mM.

The batch was incubated with mixing for 13 hours at 60 °C to release O-acetyl groups.

Potassium phosphate buffer pH 6 was added to a final concentration of 136 mM to neutralize pH and the solution was cooled to room temperature.

The solution was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate,

pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 3.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.75. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4 °C against sodium chloride.

150 mM, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 31 Preparation of Serotype 15C Conjugate for Polyvalent Study of PCV22 Using DMSO Conjugation Streptococcus pneumoniae serotype 15B polysaccharide derivative was dissolved, sized to a target molecular weight, subjected to mild base hydrolysis to release O-acetyl groups, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction, Base Hydrolysis, and Oxidation Purified Ps capsular pneumococcal serotype 15B powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 30,000 kPa/5 passes (300 bar/5 passes). The size-reduced polysaccharide solution was heated to 60°C and pH 9.4 sodium bicarbonate buffer was added to a final concentration of 50 mM.

The batch was incubated with mixing for 12 hours at 60 °C to release O-acetyl groups.

Potassium phosphate buffer pH 6 was added to a final concentration of 150 mM to neutralize pH and the solution was cooled to room temperature.

The solution was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 3.2 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.75. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 32 Preparation of Serotype 18C Conjugate for Polyvalent Studies of PCV23 (DMSO) and PCV23 (DMSO + Aq) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was reduced in size by acid hydrolysis by adding 200 mM acetic acid, incubating at 90°C for 160 minutes, then neutralizing by adding cold potassium phosphate buffer pH 7 to 400 mM.

The size-reduced polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate,

pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO of 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 3.49 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4 °C against sodium chloride.

150 mM, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 33 Preparation of Serotype 18C Conjugate for Polyvalent Study of PCV22 Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was reduced in size by acid hydrolysis by adding 200 mM acetic acid, incubating at 90°C for 160 minutes, then neutralizing by adding cold potassium phosphate buffer pH 7 to 400 mM.

The size-reduced polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO of 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 2.49 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 34 Preparation of Serotype 19A Conjugate for Polyvalent Studies of PCV23 (DMSO) and PCV23 (DMSO + Aq) Using DMSO Conjugation Polysaccharide was dissolved, chemically activated and buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.22 micron.

The polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 20 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO of 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 3.8 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.33. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.025 % (w/v) polysorbate 20, dispensed in aliquots and frozen at ≤ −60°C.

EXAMPLE 35 Preparation of Serotype 19A Conjugate for Polyvalent Study of PCV22 Using DMSO Conjugation Polysaccharide was dissolved, chemically activated and buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.22 micron.

The polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 20 hours at 22 °C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO of 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 3.8 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.33. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.025 % (w/v) polysorbate 20, dispensed in aliquots and frozen at ≤ −60°C.

EXAMPLE 36 Preparation of Serotype 19F Conjugate for Polyvalent Studies of PCV23 (DMSO), PCV23 (DMSO + Aq) and PCV22 Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 15,000 kPa/5 passes (150 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 4°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 4 hours at 4°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO of 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 2.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.2. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was filtered at 0.2 micron, then incubated at 22°C for 4.5 days.

Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The retentate batch was 0.2 micron filtered, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.025 % (w/v) polysorbate 20, dispensed in aliquots and frozen at ≤ −60°C.

EXAMPLE 37 Preparation of Serotype 22F Conjugate for Polyvalent PCV23 Study (DMSO) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 81,000 kPa/5 passes (810 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO of 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 2.3 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered with 0.5 micron pre-filter, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.025% (w/v) polysorbate 20, dispensed in aliquots and frozen at ≤ −60°C.

EXAMPLE 38 Preparation of Serotype 22F Conjugate for Polyvalent Studies of PCV22 and PCV23 (DMSO + Aq) Using Aqueous Conjugation The polysaccharide was dissolved, size reduced, chemically activated and buffer exchanged by ultrafiltration.

The purified CRM197 was then conjugated to the activated polysaccharide using the nickel chloride in the reaction mixture, and the resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water, and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular weight.

The homogenization pressure and number of passes through the homogenizer were controlled at 35,000 kPa/5 passes (350 bar/5 passes) to reduce the size to a target molecular weight.

The size-reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5M potassium phosphate, pH 7.0. The pH of the buffer selected was to improve the stability of activated polysaccharide during the conjugation reaction.

THE

Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was filtered at 0.2 micron and combined with the buffered polysaccharide solution at a mass ratio of polysaccharide to CRM197 of 0.6. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Polysaccharide and phosphate concentrations were 7.5 g/L and 100 mM, respectively.

The polysaccharide concentration was selected to control the size of the resulting conjugate.

The solution was then filtered through 0.2 micron.

Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution.

Sodium cyanoborohydride (2 mol per mol of polysaccharide repeat unit) was added.

Conjugation continued for 120 hours to maximize polysaccharide and protein consumption.

Reduction with Sodium Borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g/L, cooled to 2 to 8 °C, and filtered at 1.2 micron.

The batch was diafiltered against 100 mM potassium phosphate, pH 7.0 at 2 at 8°C using a 100 kDa NMWCO tangential flow ultrafiltration membrane.

The batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide/L and pH adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mol of polysaccharide repeat unit) was added.

Potassium phosphate 1.5M, pH 6.0 was later added.

Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The batch was 0.2 micron filtered and adjusted to a polysaccharide concentration of 1.0 g/L with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer. The batch was aliquoted and frozen at ≤ −60 °C.

EXAMPLE 39 Preparation of Serotype 23B Conjugate to PCV23 (DMSO), Polyvalent Studies of PCV23 (DMSO + Aq) and PCV22 Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 40,000 kPa/5 passes (400 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO of 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 5.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kD NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 40 Preparation of Serotype 23F Conjugate for Polyvalent Studies of PCV23 (DMSO) and PCV23 (DMSO + Aq) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 40,000 kPa/5 passes (400 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 5 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO of 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 2.1 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.25. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) polysorbate 20, dispensed in aliquots and frozen at ≤ −60°C.

EXAMPLE 41 Preparation of Serotype 23F Conjugate for Polyvalent Study of PCV22

Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer changed by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 40,000 kPa/5 passes (400 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 4 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO of 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 2.1 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.25. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 3 hours at 22 °C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kDa NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered, then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) polysorbate 20, dispensed in aliquots and frozen at ≤ −60°C.

EXAMPLE 42 Preparation of Serotype 24F Conjugate for Polyvalent Studies of PCV23 (DMSO) and PCV23(DMSO + Aq) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was reduced in size by acid hydrolysis by adding 200 mM acetic acid, incubating at 80°C for 150 minutes, then neutralizing by adding cold potassium phosphate buffer pH 7 to 400 mM.

The size-reduced polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 2 mg Ps/mL with a sucrose concentration of 10% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide solution was spiked with sodium chloride to a final concentration of 10 mM.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 1.4 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kD NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 43 Preparation of Serotype 24F Conjugate for Polyvalent Study of PCV22 Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was reduced in size by acid hydrolysis by adding 200 mM acetic acid, incubating at 80°C for 150 minutes, then neutralizing by adding cold potassium phosphate buffer pH 7 to 400 mM.

The size-reduced polysaccharide was concentrated and diafiltered against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 2 mg Ps/mL with a sucrose concentration of 10% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide solution was spiked with sodium chloride to a final concentration of 25 mM.

Polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 1.4 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.5. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kD NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 44 Preparation of Serotype 33F Conjugate for Polyvalent Study of PCV23 (DMSO) Using DMSO Conjugation The polysaccharide was dissolved, sized to a target molecular weight, chemically activated and the buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular mass of Ps.

The homogenization pressure and number of passes through the homogenizer were controlled at 51,000 kPa/5 passes (510 bar/5 passes). The size-reduced polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 2.5 g Ps/L and a mass ratio of polysaccharide to CRM197 of 1.75. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Sodium cyanoborohydride (1 mol per mol of polysaccharide repeat unit) was added, and conjugation proceeded at 22°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 22°C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 45 Preparation of Serotype 33F Conjugate for Polyvalent Studies of PCV22 and PCV23 (DMSO + Aq) Using Aqueous Conjugation The polysaccharide was dissolved, size reduced, chemically activated and buffer exchanged by ultrafiltration.

The purified CRM197 was then conjugated to the activated polysaccharide using the nickel chloride in the reaction mixture, and the resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide Size Reduction and Oxidation Purified pneumococcal capsular polysaccharide powder was dissolved in water, and filtered at 0.45 micron.

The dissolved polysaccharide was homogenized to reduce the molecular weight.

The homogenization pressure and number of passes through the homogenizer were controlled at 35,000 kPa/5 passes (350 bar/5 passes) to reduce the size to a target molecular weight.

The size-reduced polysaccharide was then concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 The oxidized polysaccharide solution was mixed with water and 1.5M potassium phosphate, pH 7.0. The pH of the buffer selected was to improve the stability of activated polysaccharide during the conjugation reaction.

Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was filtered at 0.2 micron and combined with the buffered polysaccharide solution at a mass ratio of polysaccharide to CRM197 of 0.7 . The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Polysaccharide and phosphate concentrations were 6.5 g/L and 100 mM, respectively.

The polysaccharide concentration was selected to control the size of the resulting conjugate.

The solution was then filtered through 0.2 micron.

Nickel chloride was added to approximately 2 mM using a 100 mM nickel chloride solution.

Sodium cyanoborohydride (2 mol per mol of polysaccharide repeat unit) was added.

Conjugation continued for 96 hours to maximize polysaccharide and protein consumption.

Reduction with Sodium Borohydride After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g/L, cooled to 2 to 8 °C, and filtered at 1.2 micron.

The batch was diafiltered against 100 mM potassium phosphate, pH 7.0 at 2 at 8°C using a 100 kDa NMWCO tangential flow ultrafiltration membrane.

The batch, recovered in the retentate, was then diluted to approximately 2.0 g polysaccharide/L and pH adjusted with the addition of 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mol of polysaccharide repeat unit) was added. 1.5M potassium phosphate, pH 6.0 was later added.

Final Filtration and Product Storage The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4°C using a 300 kDa NMWCO tangential flow ultrafiltration membrane.

The batch was 0.2 micron filtered and adjusted to a polysaccharide concentration of 1.0 g/L with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 buffer. The batch was aliquoted and frozen at ≤ −60 °C.

EXAMPLE 46 Preparation of Serotype 35B Conjugate for Polyvalent Studies of PCV23 (DMSO) and PCV23 (DMSO + Aq) Using DMSO Conjugation Polysaccharide was dissolved, chemically activated and buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide solution was spiked with sodium chloride to a final concentration of 20 mM.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 6.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 3.0. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Conjugation proceeded at 34°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 34°C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kD NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE 47 Preparation of Serotype 35B Conjugate for Multipurpose Study of

PCV22 Using DMSO Conjugation The polysaccharide was dissolved, chemically activated and buffer exchanged by ultrafiltration.

Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO.

The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below.

The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration.

Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes.

Polysaccharide oxidation Purified pneumococcal capsular Ps powder was dissolved in water and filtered at 0.45 micron.

The dissolved polysaccharide was concentrated and diafiltered against water using a 10 kDa NMWCO tangential flow ultrafiltration membrane.

The polysaccharide solution was then adjusted to 22°C and pH 5 with a sodium acetate buffer to minimize polysaccharide size reduction due to activation.

Polysaccharide activation was initiated with the addition of a 100 mM sodium metaperiodate solution.

The oxidation reaction proceeded for 2 hours at 22°C.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 followed by diafiltration against water using a 5 kDa NMWCO tangential flow ultrafiltration membrane.

Ultrafiltration was conducted at 2 to 8°C.

Conjugation of Polysaccharide to CRM197 Purified CRM197, obtained by expression in Pseudomonas fluorescens as previously described (WO 2012/173876 A1), was diafiltered against 2 mM phosphate, pH 7.2 buffer using a tangential flow ultrafiltration membrane of NMWCO 5 kDa and filtered at 0.2 micron.

The activated polysaccharide was formulated by lyophilization at 6 mg Ps/mL with a sucrose concentration of 5% w/v.

CRM197 was formulated by lyophilization at 6 mg Pr/ml with sucrose concentration of 1% w/v.

The formulated Ps and CRM197 solutions were individually lyophilized.

The lyophilized Ps and CRM197 materials were individually redissolved in equal volumes of DMSO.

The polysaccharide solution was spiked with sodium chloride to a final concentration of 20 mM.

The polysaccharide and CRM197 solutions were mixed to obtain a polysaccharide concentration of 6.0 g Ps/L and a mass ratio of polysaccharide to CRM197 of 3.0. The mass ratio was selected to control the ratio of polysaccharide to CRM197 in the resulting conjugate.

Conjugation proceeded at 34°C.

Reduction with Sodium Borohydride Sodium borohydride (2 mol per mol of polysaccharide repeat unit) was added after the conjugation reaction and incubated for 1 hour at 34°C.

The batch was diluted in 150 mM sodium chloride with approximately 0.025% (w/v) polysorbate 20 at approximately 4°C.

Potassium phosphate buffer was then added to neutralize the pH.

The batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride, 25 mM potassium phosphate pH 7, using a 30 kD NMWCO tangential flow ultrafiltration membrane.

Final filtration and product storage The batch was then concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 at 4°C using a membrane of 300 kDa NMWCO tangential flow ultrafiltration.

The retentate batch was 0.2 micron filtered (with 0.5 micron pre-filter), then diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0 with 0.015 % (w/v) of polysorbate 20, dispensed in aliquots and frozen at ≤ -60°C.

EXAMPLE Y Preparation of Serotypes for Polyvalent Study of PCV24 Using DMSO Conjugation Conjugates were prepared for the study of PCV24 using methods similar to those described in previous Examples. For each serotype, the polysaccharide was dissolved, chemically activated and the buffer exchanged by ultrafiltration. Activated polysaccharide and purified CRM197 were individually lyophilized and redissolved in DMSO. The re-dissolved polysaccharide and CRM197 solutions were then combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before a final 0.2 micron filtration. Various process parameters at each step, such as pH, temperature, concentration and time were controlled to yield conjugates with desired attributes. Differences from previous Examples may include: homogenization pressure and number of passes, oxidation time, concentration of polysaccharide and sucrose for lyophilization, concentration of polysaccharide during conjugation, mass ratio of polysaccharide to CRM197, and salt concentration in the conjugation reaction . EXAMPLE 48 Formulation of Pneumococcal Conjugate Vaccines Individual pneumococcal polysaccharide-protein conjugates prepared using different chemicals as described in Examples, supra, were used for the formulation of pneumococcal conjugate vaccines.

8-, 15-, 22-, 23- and 24-valent.

PCV8/APA vaccine drug product is prepared by individual conjugation of CRM197 protein to Pneumococcal Polysaccharide (PnPs) Types (-8, -10A, -12F, -15A, -15C, -23B, -24F and -35B) using reductive amination in an aprotic solvent (also called DMSO chemical) and formulated in 20 mM L-Histidine pH 5.8 and 150 mM NaCl, 0.1 % w/v polysorbate 20 (PS-20) and 250 µg [ Al]/mL in the form of Aluminum Phosphate Adjuvant as the adjuvant at 4 µg/mL, each serotype for a total polysaccharide concentration of 32 µg/mL.

PCV15/APA vaccine drug product is prepared by individual conjugation of CRM197 protein to Pneumococcal Polysaccharide (PnPs) Types (-6A, -6B, -7F, -9V, -18C, -19A, -19F, -23F) using reductive amination in an aprotic solvent (also called a DMSO chemical) or for Types -1, -3, -4, -5, -14, -22F and -33F using reductive amination in a protic solvent (also called a chemical aqueous) and formulated in 20 mM L-Histidine pH 5.8 and 150 mM NaCl, 0.2 % w/v polysorbate-20 (PS-20) and 250 µg [Al]/mL in the form of Aluminum Phosphate as the adjuvant at 4 µg/mL of each serotype (except 6B at 8 µg/mL) for a total polysaccharide concentration of 64 µg/mL.

The PCV22 vaccine drug product used to immunize mice and rabbits was prepared by individual conjugation of the CRM197 protein to Types (-1, -3, -4, -5, -6A, -6B, -7F, -9V, -10A , -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F and -35B) of Pneumococcal Polysaccharide (PnPs) using reductive amination in protic and aprotic solutions (DMSO/Aqueous “Aq”) and formulated in 20 mM L-Histidine pH 5.8 and 150 mM NaCl and polysorbate-20 (PS-20) at 0.2 % w/v and 0.8 µg/ mL, each serotype (except 6B at 1.6 µg/mL) for a total polysaccharide concentration of 18.4 µg/mL called PCV22 without adjuvant or without adj.

In another specific embodiment, the formulation is prepared with 50 µg [Al]/mL in the form of Aluminum Phosphate as the adjuvant named PCV22/APA.

PCV23 vaccine drug product is prepared by individual conjugation of CRM197 protein to Types (-1, -3, -4, -5, -6A, -6B, -7F, -8, -9V, -10A, -12F , -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F and -35B) of Pneumococcal Polysaccharide (PnPs) using reductive amination in an aprotic solvent (also called DMSO chemical) and formulated in 20 mM L-Histidine pH 5.8 and 150 mM NaCl and 0.2 % w/v polysorbate-20 (PS-20) at 4 µg/mL, each -serotype for a total polysaccharide concentration of 92 µg/mL, designated as PCV23 without adjuv.

In another specific embodiment, the formulation is prepared with 250 µg [Al]/mL in the form of Aluminum Phosphate Adjuvant as the adjuvant named PCV23/APA.

In another embodiment, the PCV23 vaccine drug product is prepared by individual conjugation of the CRM197 protein to Pneumococcal Polysaccharide Types (-1, -3, -4, -5, -9V, -14, -22F, and -33F) ( PnPs) using reductive amination in a protic solvent (also called aqueous chemical) and conjugating the CRM197 protein to Types (-6A, -6B, -7F, -8, -10A, -12F, -15A, -15C, -18C, -19A, -19F, -23B, -23F, -24F and -35B) of Pneumococcal Polysaccharide (PnPs) using reductive amination in an aprotic solvent (also called DMSO chemical). The vaccine drug product is formulated in 20 mM L-Histidine pH 5.8 and 150 mM NaCl and 0.1 % w/v Polysorbate-20 (PS-20) with 0.01 % CarboxyMethyl Cellulose (CMC) at 4 µg/mL, each serotype (except 6B at 8 µg/mL) for a total polysaccharide concentration of 96 µg/mL and prepared with 250 µg [Al]/mL in the form of Aluminum Phosphate Adjuvant as the adjuvant and named PCV23 (DMSO + Aq)/APA.

The PCV24 multivalent immunogenic composition is prepared by individual conjugation of the CRM197 protein to S. pneumoniae polysaccharide (PnPs) serotypes -1, -3, -4, -5, -6A, -6B, -7F, -8, - 9V, -10A, -11A, -12F, -14, -15A, -15C, -18C, -19A, -19F, -22F, -23B, -23F, -24F, -33F and -35B using reductive amination in an aprotic solvent (also called DMSO chemical) and formulated in 20 mM L-Histidine pH 5.8, 150 mM NaCl and 0.1 % w/v Polysorbate-20 (PS-20) 4 µg/mL or 8 µg/mL of each polysaccharide serotype to a total polysaccharide concentration of 96 µg/mL or 192 µg/mL, respectively, and called “PCV24 without adj”. In another specific embodiment, the PCV24 multivalent immunogenic composition is prepared in 20 mM L-Histidine pH 5.8, 150 mM NaCl and 0.2 % w/v Polysorbate-20 (PS-20) at 4 µg/mL of each polysaccharide serotype to a total polysaccharide concentration of 96 µg/ml further comprising 250 µg [Al]/ml in the form of Aluminum Phosphate Adjuvant.

This is called “PCV24/APA”. The required volume of bulk conjugates needed to obtain the target concentration of individual serotypes was calculated based on the batch volume and the concentration of individual concentrations of the bulk polysaccharide.

The individual conjugates were added to a solution of histidine, sodium chloride and Polysorbate-20 (PS-20) to produce a 2X to 4X conjugate blend.

The formulation vessel containing the conjugate blend is mixed using a magnetic stir bar, sterile filtered in another vessel.

The sterile filtered 2x to 4x blend is added to another container containing Aluminum Phosphate Adjuvant or diluted with saline to obtain the desired target total polysaccharide, excipient and APA adjuvant concentrations (if required). The formulations are then filled into ampoules or glass syringes and stored at 2 to 8°C.

EXAMPLE 49

PCV22 Immunogenicity and Functional Antibody in Mice Young female Balb/c mice (6 to 8 weeks old, n = 10/group) were immunized with 0.1 mL of a 22-valent pneumococcal conjugate vaccine (PCV22/APA or PCV22 without adjuvant) on day 0, day 14 and day 28. PCV22 was dosed at 0.08 µg of each pneumococcal polysaccharide (1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C , 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B) and 6B at 0.16 µg and all were conjugated to CRM197 without adjuvant or with 5 µg aluminum phosphate adjuvant (APA) per immunization.

Mice were observed at least daily by trained animal care staff for any signs of illness or discomfort.

Vaccine formulations in mice were found to be safe and well tolerated, as no vaccine-related adverse events were observed.

On day 52 mice were challenged intratracheally with Streptococcus pneumoniae serotype 24F.

Exponential phase cultures of S. pneumoniae were centrifuged, washed and suspended in sterile PBS.

Mice were anesthetized with isoflurane before challenge. 10 5 cfu of S. pneumoniae in 0.1 mL of PBS was placed in the throats of mice suspended vertically by their incisors.

Aspiration of bacteria was induced by gently pulling the tongue out and covering the nostrils.

Mice were weighed daily and euthanized if weight loss exceeded 20% of starting weight.

Blood was collected at 24 hours, 48 hours and 72 hours to assess for bacteremia.

Mice were observed at least twice daily by trained animal care staff for any signs of illness or discomfort.

All animal experiments were performed in strict accordance with the recommendations in the National Institutes of Health Guide to the Care and Use of Laboratory Animals.

The mouse experimental protocol was approved by the Institutional

Animal Care and Use at Merck & Co., Inc.

Mouse sera were evaluated for IgG immunogenicity using a multiplexed electrochemiluminescence (ECL) assay. This assay was developed for use with mouse serum based on the human assay described by Marchese et al.[3] using technology developed by MesoScale Discovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, MD) utilizing a SULFO-TAG™ tag that emits light upon electromagnetic stimulation.

SULFO-TAG™ labeled anti-mouse IgG was used as the secondary antibody to test mouse serum samples.

Functional antibody was determined by multiplexed opsonophagocytic assays (MOPA) based on protocols described previously at www.vaccine.uab.edu and Opsotiter® 3 software owned and licensed by the University of Alabama Research Foundation (UAB)[1, 2 ]. Mouse sera were pooled for each group and tested in multiplexed electrochemiluminescent assays to determine antibody titers.

PCV22 generated antibody titers in mice for all serotypes after immunizations 1, 2 and 3 with the vaccine (FIGURE 1). PCV22 showed cross-reactivity to serotype 15B, as evidenced by IgG titers (FIGURE 1). PCV22 formulated with APA tends towards superior immunogenicity compared to PCV22 without adjuvant in mice at PD2 (data not shown) and PD3 (FIGURE 2). Mouse sera were pooled for each group and tested in multiplexed opsonophagocytic assays (MOPA) to determine functional antibody titers.

PCV22 generated functional antibody titers in mice that killed vaccine-type bacterial serotypes after 3 vaccine immunizations (FIGURE 3). PCV22 formulated with APA tended towards higher functional antibody titers compared to unadjuvanted PCV22 at PD2 (data not shown) and PD3 (FIGURE 4), similar to IgG titers (FIGURE 2). Mice immunized with PCV22 were protected against intratracheal challenge with 24F of S. pneumoniae (FIGURE 5). The Mantel Cox log rank test indicated that both PCV22 without adjuvant (PCV22 without adj) and PCV22/APA groups were significantly protected against challenge when compared to the naïve group (P<0.0001). Similarly, both groups of PCV22 immunized mice had little or no bacteremia, which was significantly lower when compared to the naïve group (data not shown). EXAMPLE 50 Immunogenicity of PCV22 and Functional Antibody in Rabbits Adult white New Zealand rabbits (NZWR, n=5/group) were immunized intramuscularly (IM) with 0.1 ml of a 22-valent pneumococcal conjugate vaccine (PCV22/APA or PCV22 without adjuvant) on day 0 and day 14 (alternate sides). PCV22 was dosed at 0.08 µg of each pneumococcal polysaccharide (1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F , 33F, 35B) and 6B at 0.16 µg and all conjugated to CRM197 and without adjuvants or formulated with 5 µg APA per immunization.

Sera were collected before the start of the study (pre-immunization) and on days 14 (PD1) and 28 (PD2). NZWRS were observed at least daily by trained animal care staff for any signs of illness or discomfort.

Vaccine formulations in NZWRS were found to be safe and well tolerated, as no vaccine-related adverse events were observed.

All animal experiments were performed in strict accordance with the recommendations in the National Institutes of Health Guide to the Care and Use of Laboratory Animals.

The NZWR experimental protocol was approved by the Institutional Animal Care and Use Committees at both Merck & Co., Inc and Covance (Denver, PA). Rabbit sera were evaluated for IgG immunogenicity using a multiplexed electrochemiluminescence (ECL) assay. This assay was developed for use with rabbit serum based on the assay

[3] human described by Marchese et al. using technology developed by MesoScale Discovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, MD) utilizing a SULFO-TAG™ tag that emits light upon electromagnetic stimulation. SULFO-TAG™ labeled anti-rabbit IgG was used as the secondary antibody to test NZWR serum samples. Functional antibody was determined by multiplexed opsonophagocytic assays (MOPA) based on protocols described previously at www.vaccine.uab.edu and Opsotiter® 3 software owned and licensed by the University of Alabama Research Foundation (UAB)[1, 2 ]. Rabbit sera were individually tested in multiplexed electrochemiluminescent assays to determine antibody titers. PCV22 generated antibody titers in rabbits for all serotypes after immunizations with the vaccine (FIGURE 6). Immunization of rabbits with PCV22 also generates antibodies that bind to serotype 15B polysaccharide (FIGURE 6). There was no benefit in including APA with PCV22 in rabbits, as immunogenicity was comparable to or less than (serotype 1) PCV22 without PD2 adjuvant (FIGURE 7). Rabbit sera were individually tested in multiplexed opsonophagocytic assays (MOPA) to determine functional antibody titers. PCV22 generated functional antibody titers in rabbits that killed vaccine-type bacterial serotypes after 2 vaccine immunizations (FIGURE 8). PCV22 without adjuvant had functional antibody titers greater than PD1 for serotype 4 (data not shown) and PD2 for serotypes 1, 3 and 4 compared to PCV22 formulated with APA.

PCV22 formulated with APA did not have functional antibody titers greater than PD1 (data not shown) and PD2 for most serotypes compared to PCV22 without adjuvant (FIGURE 8). EXAMPLE 51 Immunogenicity of PCV23 in Newborn Rhesus Monkeys Newborn Rhesus monkeys (IRM, 2 to 3 months old, n=8 to 9/group) were immunized intramuscularly with 0.1 ml of a pneumococcal conjugate vaccine 23 -valent (PCV23) on days 0, 28 and 56. PCV23 was dosed at 9.6 µg of total pneumococcal polysaccharide (1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B at 0.4 µg, 6B at 0.8 µg and all conjugated to CRM197) without adjuvant or formulated with 25 µg aluminum phosphate adjuvant (APA) by immunization.

An additional group of IRMs were immunized intramuscularly with a 0.1 ml of PCV15. PCV15 was dosed at 6.4 µg of total pneumococcal polysaccharide (1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F at 0.4 µg, 6B at 0. 8 µg and all conjugated to CRM197 with 25 µg of APA per immunization) in a quadriceps and 0.1 mL of PCV8 (8, 10A, 12F, 15A, 15C, 23B, 24F and 35B at 0.4 μg and all conjugated to CRM197 with 25 µg APA per immunization) in a separate quadriceps after the same schedule as described above.

Sera were collected before the start of the study (pre-immunization, day 0) and on days 14 (PD1), 28, 42 (PD2), 56 and 70 (PD3). MRIs were observed at least daily by trained animal care staff for any signs of illness or discomfort.

All animal experiments were performed in strict accordance with the recommendations in the National Institutes of Health Guide to the Care and Use of Laboratory Animals.

The experimental protocol was approved by the

Institutional Animal Care and Use Committee at Merck & Co., Inc and the New Iberia Research Center.

Rhesus sera were evaluated for IgG immunogenicity using a multiplexed electrochemiluminescence (ECL) assay. This assay was developed for use with Rhesus sera based on the human assay described by Marchese et al. and Skinner et al [3, 4] using technology developed by MesoScale Discovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, MD) using a SULFO-TAG™ tag that emits light upon electromagnetic stimulation.

SULFO-TAG™ labeled anti-human IgG was used as the secondary antibody to test Rhesus serum samples.

IRM immunization with PCV23 generated antibody titers for all serotypes for all of the PCV23 vaccine formulations evaluated (FIGURES 9A to 9D). It should be noted that PCV23, containing polysaccharide conjugates 15A-CRM197 and 15C-CRM197, also cross-reacts to 15B, as evidenced in ECL (FIGURES 9A to 9D). PCV23 was immunogenic with a dose of vaccine in the IRMs (FIGURES 9A to 9D). The PCV23 (DMSO)/APA formulation had superior immunogenicity for serotype 22F compared to PCV23 without adjuvant to PD1, while PCV23 (DMSO+Aq)/APA had superior immunogenicity for serotype 1 and serotype 15C compared to PCV23 without adjuvant. to PD1 (FIGURE 10A). The PCV23 (DMSO)/APA formulation had superior immunogenicity for serotype 18C compared to PCV23 without PD2 adjuvant (FIGURE 10B). PCV23 (DMSO+Aq)/APA had superior immunogenicity for most serotypes (1, 3, 4, 7F, 8, 9V, 12F, 14, 15A, 15B, 15C, 18C, 19F, 22F and 23F) in compared to PCV23 without PD2 adjuvant (FIGURE 10B). PCV23 (DMSO+Aq)/APA had superior immunogenicity for serotypes 1, 4 and 9V compared to PCV23 without PD3 adjuvant (FIGURE 10C). IRMs vaccinated with PCV23 (DMSO+Aq)/APA or with a co-administration of PCV15/APA + PCV8/APA in separate limbs did not show many differences in immunogenicity to PD1, with the exception of serotype 10A which had superior immunogenicity in the co-administration group ( FIGURE 11A). However, PCV23 (DMSO+Aq)/APA vaccinated IRMs had superior PD2 immunogenicity for most serotypes 1, 3, 4, 5, 7F, 8, 9V, 14, 15A, 15B, 15C, 18C, 19F , 22F, 23B and 23F compared to co-administration of PCV15/APA + PCV8/APA (FIGURE 11B). There were no differences in immunogenicity between the two PD3 vaccines (FIGURE 11C). Evaluating the stimulatory effects of antibody, all of the PCVs generated primary immune responses to PD1 compared to pre-immune sera for all serotypes with the exception of serotype 23B in IRMs immunized with PCV23 (DMSO+Aq)/APA (FIGURE 12A). All PCVs evaluated generated significantly higher antibody titers to PD2 and PD3 when compared to pre-immune sera for all serotypes (FIGURES 12B and 12C). A second dose of PCV results in increased antibody titers from PD2 compared to PD1 for all serotypes except serotype 3 and serotype 1 in IRMs immunized with unadjuvanted PCV23, PCV23 (DMSO)/APA, and PCV15/APA+ PCV8/APA (FIGURE 12D). A third dose of PCV results in an improvement in antibody titers for most serotypes, with the exception of IRMs immunized with PCV23 (DMSO+Aq)/APA where there is a decrease in PD3 compared to PD2, suggesting that IRMs Immunized PCV23 (DMSO+Aq)/APA achieved maximal antibody response to PD2 (FIGURE 12E).

EXAMPLE Z Immunogenicity of PCV24 in White New Zealand Rabbits and Monkeys

Newborn Rhesus New Zealand white rabbits (NZWR, n = 8/group) were immunized intramuscularly with 0.1 ml of a 24-valent pneumococcal conjugate vaccine (PCV24) on days 0 and 14. PCV24 was dosed at 9, 6 µg total pneumococcal polysaccharide (1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, deOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F , 33F and 35B at 0.4 µg and all conjugated to CRM197) and formulated with aluminum phosphate adjuvant (APA, 25 µg) per immunization.

Sera were collected before the start of the study (pre-immunization, day 0) and on days 14 (PD1) and 28 (PD2). NZWRS were observed at least daily by trained animal care staff for any signs of illness or discomfort.

Newborn rhesus monkeys (IRM, 2 to 3 months old, n=5/group) were immunized intramuscularly with 0.1 mL of a 24-valent pneumococcal conjugate vaccine (PCV24) on days 0, 28, and 56. PCV24 was dosed at 9.6 µg of total pneumococcal polysaccharide (1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, deOAc15B, 18C, 19A, 19F, 22F , 23B, 23F, 24F, 33F and 35B at 0.4 µg and all conjugated to CRM197) and formulated with aluminum phosphate adjuvant (APA, 25 µg) per immunization.

Sera were collected before the start of the study (pre-immunization, day 0) and on days 14 (PD1), 28, 42 (PD2), 56 and 70 (PD3). MRIs were observed at least daily by trained animal care staff for any signs of illness or discomfort.

All animal experiments were performed in strict accordance with the recommendations in the National Institutes of Health Guide to the Care and Use of Laboratory Animals.

The experimental protocol was approved by the Institutional Animal Care and Use Committee at Merck & Co., Inc and the New Iberia Research Center.

Rhesus sera were evaluated for IgG immunogenicity using a multiplexed electrochemiluminescence (ECL) assay. This assay was developed for use with Rhesus sera based on the human assay described by Marchese et al. and Skinner et al [3, 4] using technology developed by MesoScale Discovery (a division of MesoScale Diagnostics, LLC, Gaithersburg, MD) using a SULFO-TAG™ tag that emits light upon electromagnetic stimulation.

SULFO-TAG™ labeled anti-human IgG was used as the secondary antibody for testing Rhesus serum samples and a SULFO-TAG™ labeled anti-rabbit IgG for the New Zealand white rabbit samples.

Functional antibody was determined by multiplexed opsonophagocytic assays (MOPA) based on protocols described previously at www.vaccine.uab.edu and Opsotiter® 3 software owned and licensed by the University of Alabama Research Foundation (UAB) [1, 2 ]. Immunization of NZWR with PCV24 generated antibody titers to all serotypes in the vaccine (FIGURE 13A). It should be noted that PCV24, containing polysaccharide conjugates 15A-CRM197, deOAc15B-CRM197, 6A-CRM197, 6B-CRM197 also cross-reacts to 15B and 6C, as evidenced in ECL (FIGURE 13A). NZWR sera were individually tested in multiplexed opsonophagocytic assays (MOPA) to determine functional antibody titers.

PCV24 generated functional antibody titers in NZWRs that killed all vaccine-type bacterial serotypes (FIGURE 13B). IRM immunization with PCV24 generated antibody titers for all serotypes in the vaccine (FIGURE 14A). It should be noted that PCV24, containing polysaccharide conjugates 15A-CRM197, deOAc15B-CRM197, 6A-CRM197, 6B-CRM197 also cross-reacts to 15B and 6C, as evidenced in ECL (FIGURE 14A). MRI sera were individually tested in multiplexed opsonophagocytic assays (MOPA) to determine functional antibody titers. PCV24 generated functional antibody titers on IRMs that killed vaccine-type bacterial serotypes (FIGURE 14B), with the exception of 33F, which also had lower PD3/Pre binding antibody titers on ECL. However, when PCV24/APA was evaluated in white New Zealand rabbits, PD2 33F OPA titers were more than 58-fold higher than preimmunological titers (FIGURE 13B). References

1. Caro-Aguilar I, Indrawati L, Kaufhold RM, Gaunt C, Zhang Y, Nawrocki DK, et al. Immunogenicity differences of a 15-valent pneumococcal polysaccharide conjugate vaccine (PCV15) based on vaccine dose, route of immunization and mouse strain. Vaccine, February 7, 2017;35(6): 865 to 872.

2. Burton RL, Nahm MH. Development and validation of a fourfold multiplexed opsonization assay (MOPA4) for pneumococcal antibodies. Clin Vaccine Immunol, September 2006;13(9): 1004 to 1009.

3. Marchese RD, Puchalski D, Miller P, Antonello J, Hammond O, Green T, Rubinstein LJ, Caulfield MJ, Sikkema D. Optimization and validation of a multiplex, electrochemiluminescence-based detection assay for the quantitation of serotype-specific immunoglobulin G antipneumococcal antibodies in human serum. Clin Vaccine Immunol. March 2009;16(3): 387 to 396.

4. Skinner, J.M., et al., Pre-clinical evaluation of a 15-valent pneumococcal conjugate vaccine (PCV15-CRM197) in an infant-rhesus monkey immunogenicity model. Vaccine, 2011. 29(48): pages 8,870 to 8,876. EXAMPLE 52 Materials and Methods

Free Polysaccharide Test Free polysaccharide (polysaccharide that is not conjugated to CRM197) in conjugate sample is measured by first precipitating free protein and conjugates with deoxycholate (DOC) and hydrochloric acid.

The precipitates are then filtered and the filtrates analyzed for free polysaccharide concentration by HPSEC/UV/MALS/RI.

Free polysaccharide is calculated as a percentage of total polysaccharide measured by HPSEC/UV/MALS/RI.

Free Protein Test Free polysaccharide, polysaccharide-CRM197 conjugate and free CRM197 in conjugate samples are separated by capillary electrophoresis in micellar electrokinetic chromatography (MEKC) mode. Briefly, samples are mixed with MEKC running buffer containing 25 mM borate, 100 mM SDS, pH 9.3, and separated in a preconditioned single-fused silica capillary.

Separation is monitored at 200 nm and free CRM197 is quantified with a standard curve of CRM197. Free protein results are reported as a percentage of total protein content determined by the HPSEC/UV/MALS/RI procedure.

Molecular weight and concentration analysis of conjugates using HPSEC/UV/MALS/RI assay Conjugate samples were injected and separated by high performance size exclusion chromatography (HPSEC). Detection was achieved with ultraviolet (UV), multi-angle light scattering (MALS) and refractory index (RI) detectors in series.

Protein concentration was calculated from UV280 using an extinction coefficient.

The polysaccharide concentration was deconvoluted from the RI signal (contributed by both protein and polysaccharide) using the dn/dc factors, which are the change in a refractory index of the solution with a change in reported solute concentration in mL /g.

The average molecular weight of the samples was calculated by Astra software (Wyatt Technology Corporation, Santa Barbara, CA) using the concentration and light scattering information measured across the entire sample peak.

There are multiple forms of average molecular weight values for polydispersed molecules.

For example, number average molecular weight Mn, weight average molecular weight Mw, and weight average molecular weight z Mz (Molecules, 2015, 20, 10,313 to 10,341). Unless specified, molecular weights are weight average molecular weight.

Determination of lysine consumption in conjugated protein as a measure of the number of covalent attachments between polysaccharide and carrier protein Waters AccQ-Tag (AAA) amino acid analysis was used to measure the extent of conjugation in conjugate samples.

Samples were hydrolyzed using vapor-phase acid hydrolysis on the Eldex workstation to break down carrier proteins into their component amino acids.

Free amino acids were derived using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The derived samples were then analyzed using UPLC with UV detection on a C18 column. The average protein concentration was obtained using representative amino acids other than lysine.

The consumption of lysine during conjugation (i.e. loss of lysine) was determined by the difference between the average measured amount of lysine in the conjugate and the expected amount of lysine in the starting protein.

Claims (34)

1. A multivalent immunogenic composition comprising S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, and wherein the polysaccharide-protein conjugates include polysaccharides from a group of S. pneumoniae serotypes selected from the group consisting of: a) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; b) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; c) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; d) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; e) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; f) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, DeOAc15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; g) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; h) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; i) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; j) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; k) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; l) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; m) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; n) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; o) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; p) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; q) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; r) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; s) 1, 3, 4, 5, 6A, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; t) 1, 3, 4, 5, 6B, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; u) 1, 3, 4, 5, 6C, 7F, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; v) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; w) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; x) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; y) 1, 3, 4, 5, 6A, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; z) 1, 3, 4, 5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; aa) 1, 3, 4, 5, 6C, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B; bb) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, deO-15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; cc) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; dd) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, deO-15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ee) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ff) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; gg) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, deO-15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; hh) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ii) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; jj) 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; kk) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ll) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; mm) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; nn) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; oo) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; pp) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; qq) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; rr) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ss) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; tt) 1, 3, 4, 5, 6A, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; uu) 1, 3, 4, 5, 6B, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; vv) 1, 3, 4, 5, 6C, 7F, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; ww) 1, 3, 4, 5, 6A, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; xx) 1, 3, 4, 5, 6B, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; yy) 1, 3, 4, 5, 6C, 7F, 8, 9V, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; zz) 1, 3, 4, 5, 6A, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; aaa) 1, 3, 4, 5, 6B, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39; and bbb) 1, 3, 4, 5, 6C, 7F, 8, 9V, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F, 35B and 39. The multivalent immunogenic composition of claim 1, wherein the immunogenic composition does not comprise polysaccharide-protein conjugates that have polysaccharides from any additional S. pneumoniae serotypes. The multivalent immunogenic composition of claims 1 or 2, wherein at least one of the polysaccharide-protein conjugates is formed by a conjugation reaction comprising an aprotic solvent. The multivalent immunogenic composition of claims 1 or 2, wherein each of the polysaccharide-protein conjugates is formed by a conjugation reaction comprising an aprotic solvent. The multivalent immunogenic composition of claims 3 or 4, wherein the aprotic solvent is dimethyl sulfoxide (DMSO). The multivalent immunogenic composition of any one of claims 1 to 5, wherein the carrier protein is selected from the group consisting of Outer Membrane Protein Complex (OMPC), tetanus toxoid, diphtheria toxoid, protein D and CRM197. The multivalent immunogenic composition of any one of claims 1 to 6, wherein the carrier protein is CRM197. The multivalent immunogenic composition of any one of claims 1 to 7, wherein the composition further comprises an adjuvant. The multivalent immunogenic composition of any one of claims 1 to 7, wherein the composition does not comprise an adjuvant. A method for inducing an immune response in a human patient comprising administering the multivalent immunogenic composition of any one of claims 1 to 9 to a patient. A method for inducing a protective immune response in a human patient comprising administering the multivalent immunogenic composition of any one of claims 1 to 9 to a patient. A method for inducing a protective immune response against S. pneumoniae in a human patient comprising administering the multivalent immunogenic composition of any one of claims 1 to 9 to a patient. The method of any one of claims 10 to 12, wherein the patient has previously been treated with a multivalent pneumococcal vaccine. The method of any one of claims 10 to 13, wherein the patient is 6 weeks to 17 years of age. A method for preventing pneumococcal pneumonia and/or invasive pneumococcal disease in patients 6 weeks to 17 years of age comprising administering the multivalent immunogenic composition of claim 1 to patients 6 weeks to 17 years of age. 16. A multivalent immunogenic composition comprising 23 distinct S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein each polysaccharide-protein conjugate comprises a polysaccharide from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and wherein the carrier protein is CRM197. The multivalent immunogenic composition of claim 16, wherein the immunogenic composition does not comprise polysaccharide-protein conjugates that have polysaccharides from any additional S. pneumoniae serotypes. The multivalent immunogenic composition of claim 16, wherein each of the polysaccharide-protein conjugates is formed by a conjugation reaction comprising an aprotic solvent, wherein the aprotic solvent is dimethyl sulfoxide (DMSO). The multivalent immunogenic composition of claim 16, wherein the composition comprises an adjuvant. 20. A multivalent immunogenic composition comprising 24 distinct S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein each polysaccharide-protein conjugate comprises a polysaccharide from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and wherein the carrier protein is CRM197. The multivalent immunogenic composition of claim 20, wherein the immunogenic composition does not comprise polysaccharide-protein conjugates that have polysaccharides from any additional S. pneumoniae serotypes. The multivalent immunogenic composition of claim 20, wherein each of the polysaccharide-protein conjugates is formed by a conjugation reaction comprising an aprotic solvent, wherein the aprotic solvent is dimethyl sulfoxide (DMSO). The multivalent immunogenic composition of claim 20, wherein the composition comprises an adjuvant. 24. A multivalent immunogenic composition comprising 24 distinct S. pneumoniae polysaccharide-protein conjugates, wherein each of the conjugates comprises a capsular polysaccharide of an S. pneumoniae serotype conjugated to a carrier protein, wherein each polysaccharide-protein conjugate comprises a polysaccharide from S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B, respectively, and wherein the carrier protein is CRM197. The multivalent immunogenic composition of claim 24, wherein the immunogenic composition does not comprise polysaccharide-protein conjugates that have polysaccharides from any additional S. pneumoniae serotypes. The multivalent immunogenic composition of claim 24, wherein each of the polysaccharide-protein conjugates is formed by a conjugation reaction comprising an aprotic solvent, wherein the aprotic solvent is dimethyl sulfoxide (DMSO). The multivalent immunogenic composition of claim 24, wherein the composition comprises an adjuvant. 28. A multivalent immunogenic composition comprising up to 30 distinct S. pneumoniae protein-polysaccharide conjugates, wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, and wherein the up to 30 S. pneumoniae conjugates protein-polysaccharides include polysaccharides from a group of S. pneumoniae serotypes selected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A, 15C, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. The multivalent immunogenic composition of claim 28, wherein the up to 30 polysaccharide-protein conjugates additionally include one, two, three, four, five or six additional S. pneumoniae serotypes selected from 7C, 9N, 16F, 23A , 35F and 38. 30. A multivalent immunogenic composition comprising up to 30 distinct S. pneumoniae protein-polysaccharide conjugates, wherein each of the conjugates comprises a polysaccharide from an S. pneumoniae serotype conjugated to a carrier protein, and wherein the up to 30 S. pneumoniae conjugates Polysaccharide-protein include polysaccharides from a group of S. pneumoniae serotypes selected from the group consisting of: 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15A , 15B, 18C, 19A, 19F, 22F, 23B, 23F, 24F, 33F and 35B. The multivalent immunogenic composition of claim 30, wherein the up to 30 polysaccharide-protein conjugates additionally include one, two, three, four, five or six additional S. pneumoniae serotypes selected from 7C, 9N, 16F, 23A , 35F and 38. 32. The multivalent immunogenic composition of any one of claims 28 to 31, wherein the carrier protein is CRM197. The multivalent immunogenic composition of any one of claims 28 to 32, wherein each of the polysaccharide-protein conjugates is formed by a conjugation reaction comprising an aprotic solvent, wherein the aprotic solvent is dimethylsulfoxide (DMSO). The multivalent immunogenic composition of any one of claims 28 to 33, wherein the composition comprises an adjuvant.